Baclofen and acamprosate based therapy of neurological disorders

ABSTRACT

The present invention relates to combinations and methods for the treatment of neurological disorders related to glutamate excitotoxicity and Amyloid β toxicity. More specifically, the present invention relates to novel combinatorial therapies of Alzheimer&#39;s disease, Alzheimer&#39;s disease related disorders, amyotrophic lateral sclerosis, multiple sclerosis, Parkinson&#39;s disease, Huntington&#39;s disease, neuropathic pain, alcoholic neuropathy, alcoholism or alcohol withdrawal, or spinal cord injury, based on baclofen and acamprosate combination.

FIELD OF THE INVENTION

The present invention relates to combinations and methods for thetreatment of neurological diseases and disorders. More specifically, thepresent invention relates to novel combinatorial therapy of neurologicaldisorders, based on baclofen and acamprosate combination.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is the prototypic cortical dementiacharacterized by memory deficit together with dysphasia (languagedisorder in which there is an impairment of speech and of comprehensionof speech), dyspraxia (disability to coordinate and perform certainpurposeful movements and gestures in the absence of motor or sensoryimpairments) and agnosia (ability to recognize objects, persons, sounds,shapes, or smells) attributable to involvement of the corticalassociation areas. Special symptoms such as spastic paraparesis(weakness affecting the lower extremities) can also be involved [1-4].

Incidence of Alzheimer's disease increases dramatically with the age. ADis at present the most common cause of dementia. It is clinicallycharacterized by a global decline of cognitive function that progressesslowly and leaves end-stage patients bound to bed, incontinent anddependent on custodial care. Death occurs, on average, 9 years afterdiagnosis [5]

United Nations population projections estimate that the number of peopleolder than 80 years will approach 370 million by the year 2050.Currently, it is estimated that 50% of people older than age 85 yearsare afflicted with AD. Therefore, more than 100 million people worldwidewill suffer from dementia in 50 years. The vast number of peoplerequiring constant care and other services will severely affect medical,monetary and human resources [6].

Memory impairment is the early feature of the disease and involvesepisodic memory (memory for day-today events). Semantic memory (memoryfor verbal and visual meaning) is involved later in the disease. Bycontrast, working memory (short-term memory involving structures andprocesses used for temporarily storing and manipulating information) andprocedural memory (unconscious memory that is long-term memory of skillsand procedure) are preserved until late. As the disease progresses, theadditional features of language impairment, visual perceptual andspatial deficits, agnosias and apraxias emerge.

The classic picture of Alzheimer's disease is sufficientlycharacteristic to allow identification in approximately 80% of cases[7]. Nevertheless, clinical heterogeneity does occur and this is notonly important for clinical management but provides further implicationof specific medication treatments for functionally different forms [8].

The pathological hallmark of AD includes amyloid plaques containingbeta-amyloid (Abeta), neurofibrillary tangles (NFT) containing Tau andneuronal and synaptic dysfunction and loss [9-11]. For the last decade,two major hypotheses on the cause of AD have been proposed: the “amyloidcascade hypothesis”, which states that the neurodegenerative process isa series of events triggered by the abnormal processing of the AmyloidPrecursor Protein (APP) [12], and the “neuronal cytoskeletaldegeneration hypothesis” [13], which proposes that cytoskeletal changesare the triggering events. The most widely accepted theory explaining ADprogression remains the amyloid cascade hypothesis [14-16] and ADresearchers have mainly focused on determining the mechanisms underlyingthe toxicity associated with Abeta proteins. Microvascular permeabilityand remodeling, aberrant angiogenesis and blood-brain barrier (BBB)breakdown have been identified as key events contributing to the APPtoxicity in the amyloid cascade [17]. On the contrary, Tau protein hasreceived much less attention from the pharmaceutical industry thanamyloid, because of both fundamental and practical concerns. Moreover,synaptic density change is the pathological lesion that bettercorrelates with cognitive impairment than the two others. Studies haverevealed that the amyloid pathology appears to progress in aneurotransmitter-specific manner where the cholinergic terminals appearmost vulnerable, followed by the glutamatergic terminals and finally bythe GABAergic terminals [11]. Glutamate is the most abundant excitatoryneurotransmitter in the mammalian nervous system. Under pathologicalconditions, its abnormal accumulation in the synaptic cleft leads toglutamate receptor overactivation [18]. Abnormal accumulation ofglutamate in synaptic clefts leads to the overactivation of glutamatereceptors that results in pathological processes and finally in neuronalcell death. This process, named excitotoxicity, is commonly observed inneuronal tissues during acute and chronic neurological disorders.

It is becoming evident that excitotoxicity is involved in thepathogenesis of multiple disorders of various etiology such as: spinalcord injury, stroke, traumatic brain injury, hearing loss, alcoholismand alcohol withdrawal, alcoholic neuropathy, or neuropathic pain aswell as neurodegenerative diseases such as frontotemporal dementia,multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis,Parkinson's disease, and Huntington's disease [19-21]. The developmentof efficient treatment for these diseases remains a major public healthissue due to their incidence as well as lack of curative treatments.

Two kinds of medication are used for improving or slowing down symptomsof AD which lay on some acetylcholinesterase modulators and a blocker ofNMDA glutamate receptors, memantine [22, 23].

NMDAR antagonists that target various sites of this receptor have beentested to counteract excitotoxicity. Uncompetitive NMDAR antagoniststarget the ion channel pore, thus reducing calcium entry intopostsynaptic neurons. Some of them reached the approval status. As anexample, memantine is currently approved in moderate to severeAlzheimer's disease. It is clinically tested in other indications thatinclude a component of excitotoxicity such as alcohol dependence (phaseII), amyotrophic lateral sclerosis (phase III), dementia associated withParkinson's disease (phase II), epilepsy, Huntington's disease (phaseIV), multiple sclerosis (phase IV), Parkinson's disease (phase IV) andtraumatic brain injury (phase IV). This molecule is however of limitedbenefit to most Alzheimer's disease patients, because it has only modestsymptomatic effects. Another approach in limiting excitotoxicityconsists of inhibiting the presynaptic release of glutamate. Riluzole,currently approved in amyotrophic lateral sclerosis, showed encouragingresults in ischemia and traumatic brain injury models [24-26]. It is atpresent tested in phase II trials in early multiple sclerosis,Parkinson's disease (does not show any better results than placebo) aswell as spinal cord injury. In 1995, the drug reached orphan drug statusfor the treatment of amyotrophic lateral sclerosis and in 1996 for thetreatment of Huntington's disease. The use of NMDA receptor antagonistssuch as memantine, felbamate, acamprosate and MRZ 2/579 for treatingdepression has also been suggested in US2010076075.

WO2009133128, WO2009133141, WO2009133142 and WO2011054759 disclose drugcombinations for use in the treatment of AD.

Despite active research in this area, there is still a need foralternative or improved efficient therapies for neurological disordersand, in particular, neurological disorders which are related toglutamate and/or amyloid beta toxicity. The present invention providesnew treatments for such neurological diseases of the central nervoussystem (CNS) and the peripheral nervous system (PNS).

SUMMARY OF INVENTION

It is an object of the present invention to provide new therapeuticmethods and compositions for treating neurological disorders. Moreparticularly, the invention relates to compositions and methods fortreating neurological disorders related to glutamate and/or amyloid beta(Aβ) toxicity, based on a combination of baclofen and acamprosate.

The invention stems, inter alia, from the unexpected discovery, by theinventors, that the combination of baclofen and acamprosate providessubstantial and unexpected benefit to patients with Alzheimer's disease.Moreover, the inventors have surprisingly discovered that thiscombination provides substantial and unexpected protection of neuronalcells against various injuries encountered in neurological disordersincluding glutamate toxicity. Thus, this combination of baclofen andacamprosate constitutes an efficient treatment for patients sufferingfrom, predisposed to, or suspected to suffer from neurologicaldisorders. The inventors have found that combinations of the inventionare efficient in counteracting the toxic cellular effects of Aβ peptidesand in correcting the cognitive impairments in relation with suchtoxicity. The invention further demonstrates that baclofen andacamprosate administration does improve memory functions in agedsubjects as well as cognitive functions, in particular by correctingcognition-related electro-physiological features in mild AD patients.

An object of this invention therefore relates to compositions comprisinga combination of baclofen and acamprosate for use in the treatment of aneurological disorder, particularly AD and related disorders, multiplesclerosis (MS), amyotrophic lateral sclerosis (ALS), Parkinson's disease(PD), neuropathies (for instance neuropathic pain or alcoholicneuropathy), frontotemporal dementia (FTD), alcoholism or alcoholwithdrawal, Huntington's disease (HD) and spinal cord injury.

The compositions of the invention may contain baclofen and acamprosateas the only active ingredients. Alternatively, the compositions maycomprise additional active ingredient(s). In this regard, a furtherobject of this invention relates to a composition comprising acombination of baclofen, acamprosate, and at least one third compoundselected from sulfisoxazole, methimazole, prilocaine, dyphylline,quinacrine, carbenoxolone, aminocaproic acid, cabergoline,diethylcarbamazine, cinacalcet, cinnarizine, eplerenone, fenoldopam,leflunomide, levosimendan, sulodexide, terbinafine, zonisamide,etomidate, phenformin, trimetazidine, mexiletine, ifenprodil,moxifloxacin, bromocriptine or torasemide, for use in the treatment ofneurological disorders in a subject in need thereof.

As it will be further disclosed in the present application, thecompounds in a combinatorial therapy of the invention may beadministered simultaneously, separately, sequentially and/or repeatedlyto the subject.

The invention also relates to any pharmaceutical composition per secomprising a combination of at least two compounds as defined above.

The compositions of the invention typically further comprise one orseveral pharmaceutically acceptable excipients or carriers. Also, thecompounds as used in the present invention may be in the form of a salt,hydrate, ester, ether, acid, amide, racemate, or isomer. They may alsobe in the form of sustained-release formulations. Prodrugs orderivatives of the compounds may be used as well.

In a preferred embodiment, a compound is used as such or in the form ofa salt, hydrate, ester, ether or sustained release form thereof. Aparticularly preferred salt for use in the present invention isacamprosate calcium.

In another preferred embodiment, a prodrug or derivative is used.

A further object of this invention is a method of preparing apharmaceutical composition, the method comprising mixing baclofen andacamprosate, in a pharmaceutically acceptable excipient or carrier.

Another object of this invention relates to a method for treating aneurological disorder in a mammalian subject in need thereof, preferablya human subject in need thereof, the method comprising administering tosaid subject an effective amount of a combination of the invention.

A further object of this invention relates to a method for treatingAlzheimer's disease (AD) or a related disorder in a mammalian subject inneed thereof, preferably a human subject in need thereof, the methodcomprising administering to said subject an effective amount of acombination of the invention.

A preferred object of this invention relates to a method for treating aneurological disorder in a mammalian subject in need thereof, preferablya human subject in need thereof, the method comprising simultaneously,separately or sequentially administering to said subject an effectiveamount of baclofen and acamprosate.

A particular object of this invention relates to a method of treatingFrontotemporal Dementia (FTD) in a mammalian subject in need thereof,preferably a human subject in need thereof, the method comprisingadministering to said subject an effective amount of a combination ofthe invention.

A more preferred object of this invention relates to a method fortreating AD or a related disorder in a mammalian subject in needthereof, preferably a human subject in need thereof, the methodcomprising simultaneously, separately or sequentially administering tosaid subject an effective amount of baclofen and acamprosate.

Another particular object of this invention relates to a method oftreating Age Associated Memory Impairment (AAMI) in a mammalian subjectin need thereof, preferably a human subject in need thereof, the methodcomprising administering to said subject an effective amount of acombination of the invention.

The invention may be used for treating a neurological disorder in anymammalian subject, preferably in any human subject, at any stage of thedisease. As it will be disclosed in the examples, the compositions ofthe invention are able to ameliorate the pathological condition of saidsubjects.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Validation of the experimental model of human beta-amyloidtoxicity on endothelial cells used for drug screening. One hour of VEGFpre-treatment at 10 nM significantly protected the capillary networkfrom this amyloid injury (+70% of capillary network compared to amyloidintoxication).

FIGS. 2A-2C: Effect of baclofen (BCL) and acamprosate (ACP) combinationtherapy on the total length of capillary network in beta-amyloidintoxicated HBMEC cultures. The human amyloid peptide (Aβ₁₋₄₂ 2.5 μM)produces a significant intoxication, above 40%, compared tovehicle-treated cells. This intoxication is significantly prevented bythe combination of acamprosate and baclofen (FIG. 2A) whereas, at thoseconcentrations, acamprosate (FIG. 2B) and baclofen (FIG. 2C) alone haveno significant effect on intoxication.

: p<0.05, significantly different from Aβ₁₋₄₂ intoxication; *: p<0.05,significantly different from vehicle; “ns” no significant effect(ANOVA+Dunnett Post-Hoc test).

FIG. 3: Effect of baclofen (BCL) and terbinafine (TBN) combinationtherapy on the total length of capillary network in beta-amyloidintoxicated HBMEC cultures. The human amyloid peptide (Aβ₁₋₄₂ 2.5 μM)produces a significant intoxication, above 40%, compared tovehicle-treated cells. This intoxication is prevented by the combinationof terbinafine and baclofen. *: p<0.05: significantly different fromcontrol (no intoxication).

FIG. 4: Validation of the experimental model of human beta amyloidtoxicity on neuronal cells used for drug screening. One hour ofEstradiol (150 nM) or BDNF (50 ng/mL) pre-treatment significantlyprotected the neurons from this amyloid injury (−94%), which isconsidered as a positive control for neuroprotection. *: p<0.05,significantly different from control (no intoxication);

: p<0.05, significantly different from Aβ₁₋₄₂ intoxication.

FIGS. 5A-5C: Effect of acamprosate (ACP) and baclofen (BCL) combinationtherapy on LDH release in human Aβ₁₋₄₂ toxicity on rat primary corticalcells. The human amyloid peptide (Aβ₁₋₄₂ 10 μM) produces a significantintoxication compared to vehicle-treated neurons. This intoxication issignificantly prevented by the combination of acamprosate and baclofen(FIG. 5A) whereas, at those concentrations, acamprosate (FIG. 5B) andbaclofen (FIG. 5C) alone have no significant effect on intoxication.

: p<:0.05, significantly different from Aβ₁₋₄₂ intoxication; *: p<:0.05,significantly different from vehicle; “ns” no significant effect.(ANOVA+Dunnett Post-Hoc test).

FIG. 6: Effect of cinacalcet (CNC) and sulfisoxazole (SFX) combinationtherapy on LDH release in human Aβ₁₋₄₂ toxicity on rat primary corticalcells. The human amyloid peptide (Aβ₁₋₄₂ 10 μM) produces a significantintoxication compared to vehicle-treated neurons. This intoxication isprevented by the combination of cinacalcet and sulfisoxazole. *: p<0.05,significantly different from vehicle (ANOVA+Dunnett Post-Hoc test).

FIG. 7: Effect of acamprosate (ACP) and baclofen (BCL) combinationtherapy on the total length of neurite network in beta-amyloidintoxicated cortical neurons. The human amyloid peptide (Aβ₁₋₄₂ 2.5 μM)produces a significant intoxication, above 15%, compared tovehicle-treated cells. This intoxication is significantly prevented bythe combination of acamprosate and baclofen whereas, at thoseconcentrations, acamprosate and baclofen alone have no significanteffect on intoxication. *: p<0.05, significantly different from Aβ₁₋₄₂intoxication; *: p<0.05, significantly different from vehicle(ANOVA+Dunnett Post-Hoc test).

FIG. 8: Effect of acamprosate and baclofen combination therapy onbehaviour as defined by Y-maze test. The amyloid peptide produces asignificant decrease in cognition as measured by percentage ofalternation (53.8% versus 73.5%). This deleterious effect issignificantly prevented (48.2% protection) by the combination ofacamprosate (0.2 mg/kg bid) and baclofen (3 mg/kg bid). ⋄: p<0.05,significantly different from Aβ₂₅₋₃₅ intoxication; *: p<0.05,significantly different from control (ANOVA+Dunnett Post-Hoc test).

FIG. 9: Effect of acamprosate and baclofen combination therapy on memoryas defined by passive avoidance (escape latency). The amyloid peptideproduces a significant decrease in memory performances as measured byescape latency compared to control. This deleterious effect issignificantly prevented (complete protection) by the combination ofacamprosate (0.2 mg/kg bid) and baclofen (3 mg/kg bid). ⋄: p<:0.05,significantly different from Aβ₂₅₋₃₅ intoxication; *: p<0.05,significantly different from control (ANOVA+Dunnett Post-Hoc test).

FIG. 10: Effect of acamprosate and baclofen combination therapy onmemory as defined by passive avoidance (step-through latency). Theamyloid peptide produces a significant decrease in memory performance asmeasured by step-through latency, above 44%, compared to control. Thisdeleterious effect is significantly prevented (78.8% protection) by thecombination of acamprosate (0.2 mg/kg bid) and baclofen (3 mg/kg bid)whereas, at those concentrations, acamprosate and baclofen alone have alower effect on intoxication. ⋄: p<0.05, significantly different fromAβ₂₅₋₃₅ intoxication; *: p<0.05, significantly different from control(ANOVA+Dunnett Post-Hoc test).

FIG. 11: Effect of acamprosate and baclofen combination therapy onneuron's density in the hippocampus. The amyloid peptide produces asignificant decrease in neuronal density as measured by the number ofneurons per millimeter in the hippocampus, above 21%, compared tocontrol. This neuronal injury is significantly prevented (63.2% ofinjured neurons are protected) by the combination of acamprosate (0.2mg/kg bid) and baclofen (3 mg/kg bid). ⋄: p<0.05, significantlydifferent from Aβ₂₅₋₃₅ intoxication; *: p<0.05, significantly differentfrom control (ANOVA+Dunnett Post-Hoc test).

FIG. 12: Effect of acamprosate and baclofen combination therapy on theblood-brain barrier (BBB) integrity. The amyloid peptide affects theblood-brain barrier, inducing a significant increase of itspermeability, above 51%, compared to control. Those damages on theblood-brain barrier are significantly prevented (66.6% of the integrityrestored) by the combination of acamprosate (0.2 mg/kg bid) and baclofen(3 mg/kg bid). ⋄: p<0.05, significantly different from Aβ₂₅₋₃₅intoxication, *. p<0.05, significantly different from control(ANOVA+Dunnett Post-Hoc test).

FIG. 13: Effect of acamprosate and baclofen combination therapy onsynaptic density as reflected by the synaptophysin concentration. Theamyloid peptide affects synapse function, inducing a significantdecrease in the synaptophysin concentration in the brain, above 34%,compared to control. Those damages on the synaptic density aresignificantly prevented (76%) by the combination of acamprosate (0.2mg/kg bid) and baclofen (3 mg/kg bid). ⋄: p<0.05, significantlydifferent from Aβ₂₅₋₃₅ intoxication, *: p<0.05, significantly differentfrom control (ANOVA+Dunnett Post-Hoc test).

FIG. 14: Protective effect of acamprosate and baclofen combinationtherapy on oxidative stress in the hippocampus. The amyloid peptideinduces a significant increase in oxidative stress in the hippocampus asmeasured by lipid peroxydation, above 59%, compared to control. Thisoxidative stress is significantly prevented (65.9%) by the combinationof acamprosate (0.2 mg/kg bid) and baclofen (3 mg/kg bid). ⋄: p<0.05,significantly different from Aβ₂₅₋₃₅ intoxication; *: p<0.05,significantly different from control (ANOVA+Dunnett Post-Hoc test).

FIG. 15: Effect of baclofen and acamprosate combination therapy againstglutamate toxicity on neuronal cortical cells. Glutamate intoxication issignificantly prevented by the combination of baclofen (400 nM) andacamprosate (1.6 nlM) whereas, at those concentrations, baclofen andacamprosate alone have no significant effect on intoxication. ⋄:p<0.001, significantly different from glutamate intoxication;(ANOVA+Dunnett Post-Hoc test).

FIG. 16: Effect of donepezil, acamprosate and baclofen combinationtherapy on behaviour and cognitive performance as defined by the Y-mazetest. The amyloid peptide produces a significant decrease in cognitionas measured by percentage of alternation (51.5% versus 71.8%). Thisdeleterious effect is significantly prevented (98% protection) by thecombination of donepezil (0.25 mg/kg/day), acamprosate (32 μg/kg bid)and baclofen (480 μg/kg bid), whereas at those concentrations the drugsalone have no significant effect. ⋄: p<0.01, significantly differentfrom Aβ₂₅₋₃₅ intoxication; *: p<0.01, significantly different fromcontrol (ANOVA+Dunnett Post-Hoc test).

FIG. 17: Comparison of protective effect of acamprosate and itsderivative homotaurine pre-treatment in human Aβ₁₋₄₂ toxicity assays onrat primary cortical cells. Aβ₁₋₄₂ produces a significant intoxicationcompared to vehicle-treated neurons. The intoxication is equallysignificantly prevented by homotaurine and acamprosate (99%, 8 nM). ⋄:p<0.0001: significantly different from Aβ₁₋₄₂ intoxication(ANOVA+Dunnett Post-Hoc test).

FIG. 18: Effect of acamprosate and baclofen combination therapy on thedevelopment of chronic progressive experimental autoimmuneencephalomyelitis (EAE) as defined by clinical score. Immunizationinduces a significant decrease in physical features as measured byclinical score. This deleterious effect is significantly prevented(p-value<0.01) by the combination of acamprosate (2 mg/kg/day) andbaclofen (30 mg/kg/day).

FIG. 19: Effect of memantine, acamprosate and baclofen combinationtherapy on behaviour and cognitive performances as defined by Y-mazetest. The amyloid peptide produces a significant decrease in cognitionas measured by percentage of alternation (57.7% versus 69.7%). Thisdeleterious effect is significantly prevented (79.2% protection) by thecombination of memantine (0.5 mg/kg/day), acamprosate (32 μg/kg bid) andbaclofen (480 μg/kg bid), whereas at those concentrations the drugsalone have no significant effect. ⋄⋄: p<0.01, ⋄: p<0.05, significantlydifferent from Aβ₂₅₋₃₅ intoxication; *: p<0.05, significantly differentfrom control (ANOVA+Dunnett Post-Hoc test).

FIG. 20: Baclofen (BCL)-acamprosate (ACP) combination protects synapseintegrity from Aβ₁₋₄₂ toxicity. Aβ₁₋₄₂ (0.3 μM) intoxication induces aloss in synaptic junctions between hippocampal neurons (almost 34%), asdetermined by quantification of colocalization of PSD93 andsynaptophysin proteins (black bar). This effect is significantlyreversed upon treatment with BCL and ACP combination (80 nM and 0.32 nMrespectively) by up to 62% (light grey bar). ***: p<0.001, significantlydifferent from Aβ intoxicated cells.

FIGS. 21A-21D: Baclofen-acamprosate combination reduces Aβ₁₋₄₂-inducedchanges within neurones in vitro. (FIG. 21A) Aβ₁₋₄₂ intoxication inducesoxidative stress as shown by the increase of 18% of MetO residues withinthe cells cultured in the presence of Aβ₁₋₄₂ compared to control.Baclofen (0.32 nM) and acamprosate (80 nM) treatment significantlylowers Aβ₁₋₄₂ induced oxidative stress as reported by a drop of 60% ofMetO residues in treated, intoxicated cells. (FIG. 21B) Aβ₁₋₄₂intoxication results in the triggering of apoptotic events as theincrease of cytoplasmic cytochrome C (Cyto C) released from mitochondria(above 150% control). Baclofen and acamprosate combination significantlyreduces by more than 30% the release of mitochondrial Cyto C inintoxicated cells. (FIG. 21C) Aβ₁₋₄₂ intoxication results in an increaseof 46% of phosphorylated Tau protein (pTau^(Ser212/Thr214)) within thecells, compared to control, non-intoxicated cells. Quantity ofphosphorylated Tau protein in Aβ₁₋₄₂ intoxicated neurons issignificantly reduced by more than 90% upon treatment with baclofen andacamprosate. (FIG. 21D) Baclofen-acamprosate combination diminishes bymore than 90% the release of glutamate in the culture medium ofAβ₁₋₄₂-intoxicated cells. Values are mean±s.e.m. ***: p<0.001 versus Aβ(ANOVA+Dunnett Post-Hoc test).

FIGS. 22A-22D: Acamprosate (ACP) acts through GABA_(A), glycine andmetabotropic glutamatergic receptor signaling. Aβ₁₋₄₂ (10 μM) produces asignificant intoxication of cells (-, black bars, up to 40% of death)compared to vehicle-treated neurons (control, white bars). Thisintoxication is efficiently prevented by acamprosate 8 nM (grey bars, upto 71% improvement). Muscimol, a GABA_(A) receptor agonist (FIG. 22A),strychnine, a glycine receptor antagonist (FIG. 22B), DHPG, a mGluR1/5agonist (FIG. 22C) and (2R,4R)-APDC, a mGluR2/3 agonist (FIG. 22D) allblock ACP protection of rat primary neuronal cells. Values aremean±s.e.m. ***: p<0.001 versus Aβ, or ACP pretreated and Aβ intoxicatedcells (ANOVA+Dunnett Post-Hoc test). “Control”: non-Aβ intoxicated nontreated cells; “-”: Aβ intoxicated cells. mGluR1/5 and mGluR2/3:metabotropic glutamate group I and II receptors respectively.

FIG. 23: Simultaneous activity on three main features of AD within givenconcentration ranges, i.e., baclofen: from 80 nM to 1 μM andacamprosate: from 320 pM to 4 nM. Baclofen-acamprosate combination hasbeen found to be neuroprotective and to improve, at the same time,synapse function (neuron plasticity) and endothelial cell function(angiogenesis). S: drug concentrations at which the combination exerts asynergistic effect in the corresponding in vitro models.

FIGS. 24A-24B: A one-month treatment with baclofen-acamprosatecombination (acamprosate 0.2 mg/kg bid and baclofen 3 mg/kg bid) iseffective in improving cognition in hAPPS_(L) mice of 8 months, whencompared to non-treated transgenic mice both during the acquisitionphase or the test phase. (FIG. 24A) On day 4 the latency observed fortreated transgenic mice is significantly different from that exhibitedby non-treated transgenic mice. (FIG. 24B) Escape latency for swim 1 andswim 2 in treated transgenic mice is significantly different fromnon-treated transgenic mice. Globally performances of all treatedtransgenic mice are better than those of non-treated transgenic mice andsimilar to those of non-transgenic animals. Values are mean±s.e.m. *:p<0.05, treated transgenic animals are different from non-treatedtransgenic animals (ANOVA+Dunnett Post-Hoc test); ^(◯◯◯): p<0.001.Performances of baclofen-acamprosate-treated animals are globallysignificantly different from performances of non-treated transgenicanimals (ANOVA+Dunnett Post-Hoc test).

FIG. 25: Baclofen-acamprosate combination efficiently reducesscopolamine-induced cognitive impairments in humans. Raw data scores inGroton Maze Learning Test (GMLT) are plotted along the vertical scale.An increase in the score corresponds to an impairment of performance inGMLT. Scopolamine (administered at H3) induces a rapid decrease incognitive performance in placebo-treated subjects (circles, dotted line)which lasts for approximately 6 hours (H9) after scopolamine injection.Baclofen-acamprosate mix (squares, grey/solid line) is efficient overthis period in reducing the deleterious effects of scopolamine oncognitive performance. A significant improvement in cognitive scores isobserved in the time window corresponding to the higher plasmaticconcentrations of baclofen and acamprosate (shaded bar below thehorizontal scale, dark: higher plasmatic concentrations, light: lowerplasmatic concentrations).

FIG. 26: ADAS-cog (Alzheimer's Disease Assessment Scale CognitiveSubscale) score evolution (change from baseline) during single blindChallenge-De-challenge-Re-challenge (CDR) clinical trial on 24 mild ADsubjects. ADAS-cog score is evaluated at the beginning and the end ofeach period during visits to the neurophysiologist (V). The efficiencyof baclofen-acamprosate mix is supported by the clear succession ofimprovement, worsening and improvement phases in correlation with thechallenge (plain line), de-challenge (dotted line) and re-challengephases (plain line). Data are obtained from the gathering of the wholedata from dose 1 and dose 2 administered patients. Cognition issignificantly improved considering the whole duration of the study (V1versus V4 score comparison, p<0.05); a significant improvement is alsonoticed at the end of the challenge phase (V1 versus V2 scorecomparison, p<0.01).

FIG. 27: Records of P300 wave all along theChallenge-De-challenge-Re-challenge (CDR) study, in a subject sufferingfrom mild AD treated with 0.4 mg acamprosate and 6 mg baclofen, eachbid. Baclofen-acamprosate combination is efficient in correcting theelectrophysiological mechanisms underlying cognitive processes in ADpatients. ERPs were recorded at each visit (V1-V4) to theneurophysiologist. Each curve is a modelling of the whole recordingsfrom the different electrodes. It clearly appears that, from V2 to V4,the P300 subcomponent waves consistently shift to the left during allthe duration of the study, thereby showing a decrease in latency. Animprovement of amplitude is also observed (Student's T-test on paireddata).

FIG. 28: A 17-week treatment with baclofen-acamprosate combinationefficiently improves working memory performances of aged mice (more than28 months). Percentage of alternation in T-maze test is significantlyimproved (by more than 50%) in treated aged mice when compared tonon-treated mice. Values are mean±s.e.m. ***p<0.001, treated agedanimals are significantly different from non-treated aged animals(ANOVA+Dunnett Post-Hoc test).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides new methods and compositions for treatingneurological disorders. The invention discloses novel drug combinationswhich allow an effective correction of such diseases and may be used inany mammalian subject.

The invention is suited for treating any neurological disorder, whethercentral or peripheral, particularly disorders wherein nerve or neuroninjuries, 3 amyloid, BBB breakdown or glutamate excitotoxicity areinvolved. Specific examples of such disorders include neurodegenerativediseases, neuropathies, spinal cord injury, and substance abuse such asalcoholism.

Definitions

“Neurodegenerative disorders” refers to diseases, such as Alzheimer'sdisease (AD) and related disorders, amyotrophic lateral sclerosis (ALS),multiple sclerosis (MS), Parkinson's disease (PD), and Huntington'sdisease (HD), encompassing a progressive loss of function and death ofneurons.

“Neuropathies” refers to conditions where nerves of the peripheralnervous system are damaged; this includes damage of the peripheralnervous system provoked by genetic factors, inflammatory disease, orchemical substance including drugs (vincristine, oxaliplatin, ethylalcohol). The treatment of neuropathies also includes the treatment ofneuropathic pain.

The invention is particularly suited for treating AD and relateddisorders. In the context of this invention, the term “related disorder”includes senile dementia of AD type (SDAT), frontotemporal dementia(FTD), vascular dementia, mild cognitive impairment (MCI) andage-associated memory impairment (AAMI).

As used herein, “treatment” includes the therapy, prevention,prophylaxis, retardation or reduction of symptoms provoked by or of thecauses of the above diseases or disorders. The term “treatment” includesin particular the control of disease progression and associatedsymptoms. The term “treatment” particularly includes i) a protectionagainst the toxicity caused by amyloid beta, or a reduction orretardation of said toxicity, and/or ii) a protection against glutamateexcitotoxicity, or a reduction or retardation of said toxicity, in thetreated subjects. The term “treatment” also designates an improvement ofcognitive symptoms or a protection of neuronal cells.

Within the context of this invention, the designation of a specific drugor compound is meant to include not only the specifically namedmolecule, but also any pharmaceutically acceptable salt, hydrate,derivative, isomer, racemate, conjugate, prodrug or derivative thereofof any chemical purity.

The terms “combination” or “combinatorial treating/therapy” designate atreatment wherein at least baclofen and acamprosate are co-administeredto a subject to cause a biological effect. In a combined therapyaccording to this invention, the at least two drugs may be administeredtogether or separately, at the same time or sequentially. Also, the atleast baclofen and acamprosate may be administered through differentroutes and protocols. As a result, although they may be formulatedtogether, the drugs of a combination may also be formulated separately.

The term “prodrug” as used herein refers to any functional derivatives(or precursors) of a compound of the present invention, which, whenadministered to a biological system, generate said compound as a resultof, e.g., spontaneous chemical reaction(s), enzyme catalysed chemicalreaction(s), and/or metabolic chemical reaction(s). Prodrugs are usuallyinactive or less active than the resulting drug and can be used, forexample, to improve the physicochemical properties of the drug, totarget the drug to a specific tissue, to improve the pharmacokinetic andpharmacodynamic properties of the drug and/or to reduce undesirable sideeffects. Some of the common functional groups that are amenable toprodrug design include, but are not limited to, carboxylic, hydroxyl,amine, phosphate/phosphonate and carbonyl groups. Prodrugs typicallyproduced via the modification of these groups include, but are notlimited to, esters, carbonates, carbamates, amides and phosphates.Specific technical guidance for the selection of suitable prodrugs isgeneral common knowledge [27-31]. Furthermore, the preparation ofprodrugs may be performed by conventional methods known by those skilledin the art. Methods which can be used to synthesize other prodrugs aredescribed in numerous reviews on the subject [28, 32-38]. For example,arbaclofen placarbil is listed in the ChemID plus Advance database(website: chem.sis.nlm.nih.gov/chemidplus/) and arbaclofen placarbil isa well-known prodrug of baclofen [39, 40].

The term “derivative” of a compound includes any molecule that isfunctionally and/or structurally related to said compound, such as anacid, amide, ester, ether, acetylated variant, hydroxylated variant, oralkylated (C1-C6) variant of such a compound. The term derivative alsoincludes structurally related compounds having lost one or moresubstituent as listed above. For example, homotaurine is a deacetylatedderivative of acamprosate. Preferred derivatives of a compound aremolecules having a substantial degree of similarity to said compound, asdetermined by known methods. Similar compounds along with their index ofsimilarity to a parent molecule can be found in numerous databases suchas PubChem (see Worldwide Website: pubchem.ncbi.nlm.nih.gov/search/) orDrugBank (see Worldwide Website: drugbank.ca/). In a more preferredembodiment, derivatives should have a Tanimoto similarity index greaterthan 0.4, preferably greater than 0.5, more preferably greater than 0.6,even more preferably greater than 0.7 with a parent drug. The Tanimotosimilarity index is widely used to measure the degree of structuralsimilarity between two molecules. Tanimoto similarity index can becomputed by software such as the Small Molecule Subgraph Detector [41,42] available online (see Worldwide Website:ebi.ac.uk/thornton-srv/software/SMSD/). Preferred derivatives should beboth structurally and functionally related to a parent compound, i.e.,they should also retain at least part of the activity of the parentdrug, more preferably they should have a protective activity against Aβor glutamate toxicity.

The term “derivative” also includes metabolites of a drug, e.g.,molecules which result from the (biochemical) modification(s) orprocessing of said drug after administration to an organism, usuallythrough specialized enzymatic systems, and which display or retain abiological activity of the drug. Metabolites have been disclosed asbeing responsible for much of the therapeutic action of the parent drug.In a specific embodiment, a “metabolite” as used herein designates amodified or processed drug that retains at least part of the activity ofthe parent drug, preferably that has a protective activity against Aβtoxicity or glutamate toxicity.

The term “salt” refers to a pharmaceutically acceptable and relativelynon-toxic, inorganic or organic salt of a compound of the presentinvention. Pharmaceutical salt formation consists of pairing an acidic,basic or zwitterionic drug molecule with a counterion to create a saltversion of the drug. A wide variety of chemical species can be used inneutralization reactions. Pharmaceutically acceptable salts of theinvention thus include those obtained by reacting the main compound,functioning as a base, with an inorganic or organic acid to form a salt,for example, salts of acetic acid, nitric acid, tartaric acid,hydrochloric acid, sulfuric acid, phosphoric acid, methane sulfonicacid, camphor sulfonic acid, oxalic acid, maleic acid, succinic acid orcitric acid. Pharmaceutically acceptable salts of the invention alsoinclude those in which the main compound functions as an acid and isreacted with an appropriate base to form, e.g., sodium, potassium,calcium, magnesium, ammonium, or choline salts. Though most of salts ofa given active principle are bioequivalents, some may have, amongothers, increased solubility or bioavailability properties. Saltselection is now a common standard operation in the process of drugdevelopment as taught by H. Stahl and C. G. Wermuth in their handbook[43].

In a preferred embodiment, the designation of a compound is meant todesignate the compound per se, as well as any pharmaceuticallyacceptable salt, hydrate, isomer, racemate, ester or ether thereof.

In a more preferred embodiment, the designation of a compound is meantto designate the compound as specifically designated per se, as well asany pharmaceutically acceptable salt thereof.

In a particular embodiment, a sustained-release formulation of thecompound is used.

Compositions and Methods of the Invention

As discussed above, the invention relates to particular drugcombinations which have a strong unexpected effect on several biologicalprocesses involved in neurological disorders. These drug combinationstherefore represent novel approaches for treating neurologicaldisorders, such as Alzheimer's disease and related disorders, multiplesclerosis, amyotrophic lateral sclerosis, Parkinson's disease,Huntington's disease, neuropathies (for instance neuropathic pain oralcoholic neuropathy), alcoholism or alcohol withdrawal, and spinal cordinjury. More specifically, the invention discloses compositions,comprising baclofen in combination with acamprosate, which provide asignificant effect in vivo on neurological disorders.

Indeed, the invention shows, in the experimental part, that combinationtherapies comprising baclofen and acamprosate can substantially improvethe condition of patients afflicted with neurological disorders. Inparticular, the inventors have surprisingly discovered that baclofen andacamprosate combinations have a strong, unexpected effect on the lengthof the capillary network, on LDH release in beta-amyloid intoxicatednervous cells as well as on the length of the neurite network, andrepresent new therapeutic approaches for AD. They have also discoveredthat baclofen-acamprosate combinations are efficient in lowering the Aβinduced oxidative stress, apoptosis, glutamate release andphosphorylated Tau accumulation in neuronal cells.

Also, the examples show that, in a combination therapy of the invention,baclofen may be effective at a dose of 80 nM or less, and acamprosatemay be effective at a dose of 1 nM or less. These results are remarkableand particularly advantageous since, at such low doses, any possibleside effects are avoided. Moreover the inventors have been able todetermine plasmatic and/or brain concentration ranges at which such acombination therapy exerts a simultaneous protective activity againstthe three aspects of Aβ toxicity in relation with AD pathogenesis, i.e.,angiogenesis, neuronal protection and neuronal plasticity. Furthermore,these combinations effectively protect neuronal cells from variousafflictions such as glutamate toxicity and oxidative stress, and preventBBB permeabilization and neuronal cell induced apoptosis, which areinvolved in several neurological disorders.

The present invention therefore proposes a novel therapy forneurological disorders, based on baclofen and acamprosate compositions.More particularly, the present invention therefore proposes a noveltherapy for Alzheimer's disease and related disorders, multiplesclerosis, amyotrophic lateral sclerosis, Parkinson's disease,Huntington's disease, neuropathies (for instance neuropathic pain oralcoholic neuropathy), alcoholism or alcohol withdrawal, and spinal cordinjury, based on baclofen and acamprosate combinations.

In this regard, in a particular embodiment, the invention relates to acomposition comprising baclofen and acamprosate.

In a further embodiment, the invention relates to a compositioncomprising baclofen and acamprosate for use in the treatment of AD,AD-related disorders, MS, PD, ALS, HD, neuropathies (for instanceneuropathic pain or alcoholic neuropathy), alcoholism or alcoholwithdrawal, or spinal cord injury.

In a further embodiment, the invention relates to the use of baclofenand acamprosate for the manufacture of a medicament for the treatment ofAD, AD-relateddisorders, MS, PD, ALS, HD, neuropathies (for instanceneuropathic pain or alcoholic neuropathy), alcoholism or alcoholwithdrawal, or spinal cord injury.

Illustrative CAS numbers for baclofen and acamprosate are provided inTable 1 below. Table 1 cites also, in a non-limitative way, commonsalts, racemates, prodrugs, metabolites or derivatives for thesecompounds used in the compositions of the invention.

TABLE 1 Class or Tanimoto Drug CAS Numbers similarity index Acamprosateand related compounds Acamprosate 77337-76-9; 77337-73-6 NA Homotaurine3687-18-1 0.73 Ethyl Dimethyl Ammonio / 0.77 Propane Sulfonate Taurine107-35-7 0.5 Baclofen and related compounds Baclofen 1134-47-0;66514-99-6; NA 69308-37-8; 70206-22-3; 63701-56-4; 63701-55-33-(p-chlorophenyl)-4- / Metabolite hydroxybutyric acid Arbaclofenplacarbil 847353-30-4 Prodrug

Specific examples of prodrugs of baclofen are given in Hanafi et al.[44], particularly baclofen esters and baclofen ester carbamates, whichare of particular interest for CNS targeting. Hence such prodrugs areparticularly suitable for compositions of this invention. Arbaclofenplacarbil as mentioned before is also a well-known prodrug and may thusbe used instead of baclofen in compositions of the invention. Otherprodrugs of baclofen can be found in the following patent applications:WO2010102071, US2009197958, WO2009096985, WO2009061934, WO2008086492,US2009216037, WO2005066122, US2011021571, WO2003077902, andWO2010120370.

Useful prodrugs for acamprosate such as pantoic acid ester neopentylsulfonyl esters, neopentyl sulfonyl ester prodrugs of acamprosate ormasked carboxylate neopentyl sulfonyl ester prodrugs of acamprosate arenotably listed in WO2009033069, WO2009033061, WO2009033054,WO2009052191, WO2009033079, US 2009/0099253, US 2009/0069419, US2009/0082464, US 2009/0082440, and US 2009/0076147.

Baclofen and acamprosate combination therapy may be used alone or may befurther combined with additional compounds. In this regard, in aparticular embodiment, the compositions of the invention may furthercomprise at least one compound selected from sulfisoxazole, methimazole,prilocaine, dyphylline, quinacrine, carbenoxolone, aminocaproic acid,cabergoline, diethylcarbamazine, cinacalcet, cinnarizine, eplerenone,fenoldopam, leflunomide, levosimendan, sulodexide, terbinafine,zonisamide, etomidate, phenformin, trimetazidine, mexiletine,ifenprodil, moxifloxacin, bromocriptine or torasemide. Illustrative CASnumbers for each of these compounds are provided in Table 2 below:

TABLE 2 DRUG NAME CAS NUMBER Aminocaproic Acid 60-32-2 Bromocriptine25614-03-3 Cabergoline 81409-90-7 Carbenoxolone 5697-56-3 Cinacalcet226256-56-0 Cinnarizine 298-57-7 Diethylcarbamazine 90-89-1 Dyphylline479-18-5 Eplerenone 107724-20-9 Etomidate 33125-97-2 Fenoldopam67227-57-0 Ifenprodil 23210-56-2 or 23210-58-4 Leflunomide 75706-12-6Levosimendan 141505-33-1 Methimazole 60-56-0 Mexiletine 5370-01-4 or31828-71-4 Moxifloxacin 354812-41-2 Phenformin 114-86-3 Prilocaine721-50-6 or 14289-31-7 or 14289-32-8 Quinacrine 83-89-6 Sulfisoxazole127-69-5 Sulodexide 57821-29-1 Terbinafine 91161-71-6 Torasemide56211-40-6 or 72810-59-4 Trimetazidine 5011-34-7 or 13171-25-0Zonisamide 68291-97-4

In a particular embodiment, the invention relates to the use of thiscombination for treating AD or a related disorder in a subject in needthereof.

In another particular embodiment, the invention relates to the use ofthis combination for treating an AD-related disorder selected fromsenile dementia of AD type (SDAT), frontotemporal dementia (FTD),vascular dementia, mild cognitive impairment (MCI) and age-associatedmemory impairment (AAMI).

A particular object of this invention relates to a method of treatingfrontotemporal dementia (FTD) in a mammalian subject in need thereof,preferably a human subject in need thereof, the method comprisingadministering to said subject an effective amount of a combination ofthe invention.

In a particular embodiment, the invention relates to the use of thiscombination for treating MS, PD, ALS, HD, neuropathies (for instanceneuropathic pain or alcoholic neuropathy), alcoholism or alcoholwithdrawal, or spinal cord injury in a subject in need thereof.

As disclosed in the examples, composition therapies using at leastbaclofen and acamprosate have a strong unexpected effect on biologicalprocesses leading to neuronal injuries. Furthermore, these combinationsalso showed in vivo a very efficient ability to correct symptoms ofneurological diseases. These combinations therefore represent novelapproaches for treating neurological disorders, such as AD, MS, ALS, PD,HD, neuropathies (for instance neuropathic pain or alcoholicneuropathy), alcoholism or alcohol withdrawal, and spinal cord injury.These compositions efficiently prevent toxicity of amyloid β (Aβ)peptide or glutamate excitotoxicity in neuronal cells. Moreparticularly, as shown in the experimental section, these compositionsare efficient at counteracting, simultaneously, the detrimental effectsof intoxication by Aβ oligomers at the synaptic, neuronal andendothelial levels. Such combination of effects is particularlyadvantageous and leads to a significant improvement of the disease bothin several in vivo models for AD and in clinical trials. Indeed, invivo, these compositions lead to an improvement of several cognitivesymptoms as well as to protection of neuronal cells.

Furthermore the experimental section shows that the above-mentionedcompositions are also efficient at i) synergistically protecting invitro neuronal cells from glutamate excitotoxicity, and ii) conferringclinical benefit in in vivo models for diseases related to glutamateexcitotoxicity.

Hence they represent novel and potent methods for treating suchdisorders.

The compositions of the invention may comprise 2, 3, 4 or 5 distinctdrugs, more preferably 2, 3 or 4 distinct drugs for combinatorialtreatment of Alzheimer's disease (AD), AD-related disorders. MS, PD,ALS, HD, neuropathies (for instance neuropathic pain or alcoholicneuropathy), alcoholism or alcohol withdrawal, or spinal cord injury ina subject in need thereof. In a preferred embodiment, the drugs of theinvention are used in combination(s) for combined, separate orsequential administration, in order to provide the most effectiveeffect.

Preferred compositions of the invention, for use in the treatment of aneurological disorder such as Alzheimer's disease (AD), AD-relateddisorders, MS, PD, ALS, HD, neuropathies (for instance neuropathic painor alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinalcord injury, comprise one of the following drug combinations, forcombined, separate or sequential administration:

-   -   baclofen and acamprosate,    -   baclofen and acamprosate and diethylcarbamazine,    -   baclofen and acamprosate and cinacalcet,    -   baclofen and acamprosate and sulfisoxazole,    -   baclofen and acamprosate and torasemide,    -   baclofen and acamprosate and ifenprodil,    -   baclofen and acamprosate and mexiletine.    -   baclofen and acamprosate and eplerenone,    -   baclofen and acamprosate and levosimendan,    -   baclofen and acamprosate and terbinafine, or    -   baclofen and acamprosate and leflunomide.

As disclosed in the experimental section, combinatorial therapies of theinvention provide substantial therapeutic and biological effects toimprove Alzheimer's disease or related disorders in both animal modeland human subjects. They induce a strong neuroprotective effect againstAB toxicity, notably through the inhibition of Aβ-induced apoptosis andoxidative stress. They also give positive results in behaviouralperformances and biochemical assays in rodents. Results show thatcompositions of the invention in vivo: (i) efficiently correct molecularpathways triggered by AD oligomers, and (ii) lead to an improvement ofneurophysiological impairments observed in diseased animals as neuronsurvival or synapse integrity. Results also show that compositions ofthe invention efficiently restore the blood-brain barrier (BBB) andprevent, retard, or lessen apoptosis triggering, which are known to beimpaired in several neurological diseases. Combinatorial therapies arealso efficient at correcting memory impairment in aged animals. Resultsfrom clinical trials in human subjects also show an activity oncognitive performances of AD patients.

Moreover, the results presented also show that the above combinationstherapies have an important synergistic neuroprotective effect againstglutamate excitotoxicity (FIG. 15), a pathway which is implicated invarious neurological diseases such as AD, MS, PD, ALS, HD, neuropathies(for instance neuropathic pain or alcoholic neuropathy), alcoholism oralcohol withdrawal, or spinal cord injury. These therapies give positiveresults in in vivo or in vitro models for these diseases.

Furthermore, the particularly high synergistic interaction observed forthese two drugs through the combinatorial treatment of the inventionallows the use of drug concentrations showing no effect when used insingle-drug treatment. Moreover, as shown in the experimental section,the baclofen and acamprosate combination causes an enhanced therapeuticbenefit in treating Alzheimer's disease compared to other therapeuticcombinations. These compositions efficiently prevent the toxic effectsof amyloid β protein or peptide on human cells and in an in vivo modeland represent novel and potent methods for treating such disorder.

An object of this invention thus also resides in a composition asdefined above for treating a neurological disorder such as AD,AD-related disorders, MS, PD, ALS, HD, neuropathies (for instancealcoholic neuropathy or neuropathic pain), alcoholism or alcoholwithdrawal, or spinal cord injury.

As indicated previously, in a combination therapy of this invention, thecompounds or drugs may be formulated together or separately, andadministered together, separately or sequentially.

A further object of this invention resides in the use of a compositionas defined above for the manufacture of a medicament for treating aneurological disorder such as Alzheimer's disease (AD), AD-relateddisorders, MS, PD, ALS, HD, neuropathies (for instance neuropathic painor alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinalcord injury.

The invention further provides a method for treating a neurologicaldisorder such as Alzheimer's disease (AD), AD-related disorders, MS, PD,ALS, HD, neuropathies (for instance neuropathic pain or alcoholicneuropathy), alcoholism or alcohol withdrawal, or spinal cord injury,comprising administering to a subject in need thereof an effectiveamount of a composition as disclosed above.

A further object of the invention is a method of treating a neurologicaldisorder such as Alzheimer's disease (AD), AD-related disorders, MS, PD,ALS, HD, neuropathies (for instance neuropathic pain or alcoholicneuropathy), alcoholism or alcohol withdrawal, or spinal cord injury,the method comprising simultaneously, separately or sequentiallyadministering to a subject in need thereof an effective amount of acomposition as disclosed above.

In a preferred embodiment, the invention relates to a method of treatinga neurological disorder such as Alzheimer's disease (AD), AD-relateddisorders, MS, PD, ALS, HD, neuropathies (for instance neuropathic painor alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinalcord injury in a subject in need thereof, comprising administeringsimultaneously, separately or sequentially to the subject an effectiveamount of baclofen and acamprosate.

The compositions of the invention typically comprise one or severalpharmaceutically acceptable carriers or excipients. Also, for use in thepresent invention, the drugs or compounds are usually mixed withpharmaceutically acceptable excipients or carriers.

In this regard, a further object of this invention is a method ofpreparing a pharmaceutical composition, the method comprising mixing theabove compounds in an appropriate excipient or carrier.

In a particular embodiment, the method comprises mixing baclofen andacamprosate in an appropriate excipient or carrier.

According to preferred embodiments of the invention, as indicated above,the compounds are used as such or in the form of a pharmaceuticallyacceptable salt, prodrug, derivative, or sustained release formulationthereof.

Although very effective in vitro and in vivo, depending on the subjector specific condition, the combination therapy of the invention mayfurther be used in conjunction, association or combination withadditional drugs or treatments beneficial to the treated neurologicalcondition in the subject.

Other therapies used in conjunction with drug(s) or drug(s)combination(s) according to the present invention may comprise one ormore drug(s) that ameliorate symptoms of Alzheimer's disease, anAD-related disorder, MS, PD, ALS, HD, neuropathies (for instanceneuropathic pain or alcoholic neuropathy), alcoholism or alcoholwithdrawal, or spinal cord injury, or drug(s) that could be used forpalliative treatment of these disorders. For instance, results also showthat the above combination therapies have an important synergisticneuroprotective effect when combined with donepezil (FIG. 16) ormemantine (FIG. 19), thereby allowing the use of low doses of saidcompounds and avoiding or lessening side effects. Thereby, illustrativetherapies which can be used with combinations of the invention aredonepezil (CAS: 120014-06-4), galantamine (CAS: 357-70-0), gabapentine(CAS: 478296-72-9; 60142-96-3), rivastigmine (CAS: 123441-03-2) ormemantine (CAS: 19982-08-2). The above CAS numbers are only given in anillustrative way, and common salts, enantiomeric forms, racemates,prodrugs, metabolites or derivatives of the above compounds should bealso considered.

In this regard, in a particular embodiment, the drug(s) or compositionsaccording to the present invention may be further combined with GinkgoBiloba extracts. Suitable extracts include, without limitation, Ginkgobiloba extracts, improved Ginkgo biloba extracts (for example enrichedin active ingredients or lessened in contaminants) or any drugcontaining Ginkgo biloba extracts.

In another particular embodiment, the drug(s) or compositions accordingto the present invention may be further combined with drugs or compoundswhich are currently under phase III clinical trial for AD. These drugsor compounds include Epigallocatechin-3-gallate, Human neutral insulin,Idalopirdine, Vanutide cridificar, Durin-Leuprolide acetate,Gantenerumab, Latrepirdine hydrochloride, Solanezumab, Masitinibmesylate, Encenicline hydrochloride, Leuco methylthioninium salt, IGIV,Lu-AE-58-054, VP4896, INM-176, R04909832, SK-PCB70M, AC-1204, andMK8931.

Furthermore, the inventors have been able to decipher the molecularmechanisms underlying the unexpected efficiency of combinations of theinvention. The results show that (i) baclofen exerts a Gaba B agonisticactivity which is essential for neuroprotective activity and (ii)acamprosate exerts a particular pattern of biological interactions(Table 3) that is essential for a neuroprotective effect in the contextof AD or AD-related disorders

TABLE 3 Group I Group II metabotropic metabotropic Gaba A Glycineglutamatergic glutamatergic receptors receptors receptors receptorsAcamprosate antagonist agonist antagonist antagonist activity* *patternof activity that has been shown necessary to afford a protective effectagainst toxicity of Aβ

As shown in the experimental section, counteracting only one of thesemechanisms of action results in an almost total abolition of theneuroprotective effect of acamprosate against Aβ toxicity (FIG. 22).Notably, the inventors show for the first time that, in the context ofAD pathogenesis, acamprosate exerts an antagonistic effect on GABA Areceptors which is essential to afford neuroprotection against Aβ.Hence, together with the agonistic action of baclofen on GABA Breceptors, the concerted action of acamprosate on the four identifiedreceptor families leads to a particularly efficient therapeutic effectas described in this whole disclosure. This concerted action on severaltargets when using only one drug thus makes acamprosate of particularinterest.

Furthermore, based on the knowledge of the pattern of action provided inthe present application, it is now possible to design and usealternative combinations of drugs in replacement of, e.g., acamprosate.Such alternative combinations should exhibit the same pattern ofbiological interaction as shown for acamprosate in Table 3 and, inparticular, they should (i) antagonize GABA A receptors and Group I andII metabotropic receptors, and (ii) lead to increased activity ofglycinic receptors.

Consequently, in a particular embodiment, this invention relates to acombination of baclofen with a drug having an antagonist activity onGABA A receptors, as well as on Group I and II metabotropic receptors,and an agonist activity on glycinic receptors.

In a particular embodiment, the invention relates to a combination ofbaclofen with a combination of drugs, said combination of drugs havingan antagonistic activity toward GABA A receptors and Group I and IImetabotropic receptors, as well as an agonistic activity toward glycinicchannels. Said combination of drugs used as an alternative toacamprosate can comprise 2, 3, 4, 5 or even 6 drugs in order to mimicthe pleiotropic activity of acamprosate. Ideally, some of the drugs usedin said composition act on several of the targets identified by theinventors. More preferably some of the drugs used in said compositionact on 2, or 3 or 4 of the targets identified by the inventors. Hence,more preferably, the combination of drugs used as an alternative toacamprosate comprises 2, 3 or 4 drugs.

An agonistic or antagonistic activity toward a receptor/channel refersto a direct action on said receptor/channel or to an indirect actionleading to the activation or inhibition of said receptor/channel.

An agonistic or antagonist activity also refers to a positive ornegative allosteric modulation, respectively.

Drugs suitable to elaborate such a combination of drugs are listed inTable 4 below with their corresponding CAS number, IUPAC name or relatedarticles, for illustrative purposes only.

TABLE 4 antagonists of group I mGluR GRM1 GRM5 AIDA 168560-79-0 ADX10059 1166398-32-8; 757949-98-7 A-794278 869802-57-3 AZD-20661403991-95-6; 934282-55-0; A-794282 869802-44-8 934338-70-2 A-841720869802-58-4 AZD6538 Raboisson et al 2012 [45] A-850002 869802-73-3AZD9272 1166398-50-0 Bay-36-7620 232605-26-4 Basimglurant 802906-73-6FTIDC Suzuki et al 2007 [46] Dipraglurant 872363-17-2 JNJ-16259685409345-29-5 Fenobam 57653-26-6; 63540-28-3 LY-367385 198419-91-9 GRN5291253291-12-1 RO0711401 714971-87-6 LY-344545 201851-20-9 YM-202074299900-83-7 Mavoglurant 543906-09-8 YM-230888 Kohara et al 2007 [47]MPEP 219911-35-0 YM-298198 748758-45-4 MRZ-8676 Dekundy et al 2011 [48]MTEP 329205-68-7; 1186195-60-7 Rufinamide 106308-44-5 SIB-175731993-01-8 SIB-1893 7370-21-0 Antagonists of group II m GluR (GRM2 and3) BCI-632 569686-87-9 BCI-1038 (N/A) Prodrug of B632 BCI-1206 (N/A)Prodrug of B632 BCI-1283 (N/A) Prodrug of B632 BCI-838(1R,2R,3R,5R,6R)-2-Amino-3-[(3,4-dichlorobenzyl)oxy]-6-fluoro-6-[(heptyloxy)carbonyl]bicyclo[3.1.0]hexane- 2-carboxylic acidRG-1578 911115-16-7 RO4491533 579482-31-8 LY-341495 201943-63-7 APICA170847-18-4 EGLU 170984-72-2 Antagonists of Gaba A receptors α₅IA215874-86-5 beta-Cce 74214-62-3 beta-Ccm 69954-48-9 beta-Cct 93835-05-3bicuculline 485-49-4; 56083-00-2 BTS-72-664(R)-7-[1-(4-chlorophenoxy)]ethyl]-1,2,4-triazolo(1,5-alpha)pyrimidinecicutoxin 505-75-9 clarithromycin 81103-11-9 DMCM 82499-00-1 FG71-4278538-74-6 FGIN-1-27 142720-24-9 FGIN-1-442-hexyl-indole-3-acetamide-N-benzene-tricarboxylic acid flumazenil78755-81-4 gabazine 104104-50-9 L-655708 Ethyl(S)-11,12,13,13a-Tetrahydro-7-methoxy-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxylate lorediplon 917393-39-6MK0777 252977-51-8 oenanthotoxin 20311-78-8 pentetrazol 54-95-5picrotoxin 124-87-8 pitrazepin 90685-01-1 PWZ-0298-chloro-3-(methoxymethyl)-5-methyl-4H-imidazo[1,5-a] [1,4]benzodiazepin-6-one R048822243,10-dichloro-9H-benzo[f]imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]diazepine RO15-3505 ethyl7-chloro-5-methyl-6-oxo-5,6-dihydro-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate Ro15-4513ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,4]-benzodiazepine-3-carboxylate RO-49385813-bromo-10-(difluoromethyl)-9H-benzo[f]imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]diazepine RU-5135 78774-26-2 RY-023(tert-butyl-8-(trimethylsilyl) acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5a] [1,4] benzodiazepine-3-carboxylate RY-024t-butyl-8-ehtynyl-(5,6-dihydro-5-methyl-6-oxo-4H-imidazo (1,5-a)[1,4]benzodiazepine-3-carboxylate RY-024t-butyl-8-ehtynyl-(5,6-dihydro-5-methyl-6-oxo-4H-imidazo (1,5-a)[1,4]benzodiazepine-3-carboxylate RY-80 ethyl8-ethynyl-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate S8510 [2-(3-isoxazolyl)-3,6,7,9-tetrahydroimidazo [4,5-d]pyrano [4,3-b] pyridine monophosphate monohydrate] sarmazenil 78771-13-8SR-42641 105537-78-8 SR-95103 96440-63-0 thiocolchicoside 602-41-5thujone 1125-12-8; 546-80-5; 471-15-8; 33766-30-2 TP003 628690-75-5US-1010 516-55-2 ZK-93423 89592-45-0 Agonists of Glycinic channelsanandamide 94421-68-8 cycloserine 68-41-7 dimethylglycine 1118-68-9dronabinol 1972-08-3 enflurane 13838-16-9 glycine 56-40-6 Halothane151-67-7 HU 210 112830-95-2 hypotaurine 300-84-5 isoflurane 26675-46-7milacemide 76990-56-2 sarcosine 203-538-6 serine 302-84-1; 56-45-1;312-84-5 sevoflurane 28523-86-6 taurine 107-35-7 trimethylglycine107-43-7; 17146-86-0 alanine 302-72-7; 56-41-7; 338-69-2 β alanine107-95-9 WIN 55212-2 131543-23-2 arachidonyl- 53847-30-6 glycerol

In a particular embodiment, said combination of drugs used as analternative to acamprosate comprises at least one compound selected fromα₅TA, beta-Cce, beta-Ccm, beta-Cct, bicuculline, BTS-72-664, cicutoxin,clarithromycin, DMCM, FG71-42, FGIN-1-27, FGIN-1-44, flumazenil,gabazine, L-655708, lorediplon, MK0777, oenanthotoxin, pentetrazol,picrotoxin, pitrazepin, PWZ-029, R04882224, RO15-3505, Ro15-4513,RO-4938581, RU-5135, RY-023, RY-024, RY-024, RY-80, S8510, sarmazenil,SR-42641, SR-95103, thiocolchicoside, thujone, TP003, US-1010, orZK-93423, which are known as (direct or indirect) antagonists or inverseagonists of the GABA A receptors.

In a preferred embodiment, said combination of drugs used as analternative to acamprosate comprises thiocolchicoside.

In another particular embodiment, antagonists or inverse agonists of theGABA A receptors of use can be, but are not limited to, one of thosedescribed in WO2011/153377, WO2012/059482, EP2457569, and WO2011/024115,which are herein incorporated by reference.

In an embodiment, antagonists of the Group I metabotropic receptors thatcan be used in a combination of drugs used as an alternative toacamprosate may be, but are not limited to, one of those disclosed inWO2004014370A2, WO2004014881A2, WO2004014902A2, WO2005080356A1,WO2005080363A1, WO2005080386A1, WO2006014185A1, WO2007021574A1,WO2007021575A2, WO2007130825A2, WO2009051556A1, WO2009054785A1,WO2009054786A1, WO2009054789A1, WO2009054790A1, WO2009054791A1,WO2009054792A1, WO2009054793 A1, WO2009054794A1, WO2010123451A1,WO02068417A2, WO2005066155A1, WO2004000316A1, WO2005080379A1,WO2005080397A2, WO2007040982A1, WO2010019100A1, WO2007130820A2,WO2007130821 A2, WO2007130822A2, WO2007130823 A2, WO2007130824A2,WO2008041075A1, WO2009054787A1, WO9926927A2, WO2004069813A1,WO2012127393 A1, WO2003047581A1, and WO2008128968, which areincorporated herein by reference.

Further antagonists of the Group I metabotropic receptors that can beused are disclosed in WO2012108831 A1 and are incorporated herein byreference.

Yet further antagonists of the Group I metabotropic receptors that canbe used are disclosed in WO2012127393A1 and are incorporated herein byreference.

Other antagonists of the Group I metabotropic receptors that can be usedare disclosed in WO2010048095A2 and are incorporated herein byreference.

Antagonists of the Group I metabotropic receptors have been the focus ofnumerous research programs in drug discovery which are summarized byJaeschke et al (2007) [50], Carroll (2008) [51] and Emmitte (2013) [52].All the antagonists of the Group I metabotropic receptors disclosed inthese reviews are incorporated herein by reference and can be consideredto be used in a combination of drugs used as an alternative toacamprosate.

In a particular embodiment, said combination of drugs used as analternative to acamprosate comprises at least one compound having anantagonistic activity for mGluR1 metabotropic receptors and/or at leastone compound having an antagonistic activity for mGluR5 metabotropicreceptors.

In a particular embodiment, said combination of drugs used as analternative to acamprosate comprises at least one compound selected fromAIDA, A-794278, A-794282, A-841720, A-850002, Bay-36-7620, FTIDC,JNJ-16259685, LY-367385, RO0711401, YM-202074, YM-230888, YM-298198, ADX10059, AZD-2066, AZD6538, AZD9272, basimglurant, dipraglurant, fenobam,GRN529, LY-344545, mavoglurant, MPEP, MRZ-8676, MTEP, rufinamide,SIB-1757, and SIB-1893, which are known as (direct or indirect)antagonists of the Group I metabotropic receptors.

In a preferred embodiment, said combination of drugs used as analternative to acamprosate comprises rufinamide.

In another preferred embodiment, said combination of drugs used as analternative to acamprosate comprises a compound selected from AIDA,A-794278, A-794282, A-841720, A-850002, Bay-36-7620, FTIDC,JNJ-16259685, LY-367385, RO0711401, YM-202074, YM-230888, YM-298198 anda compound selected from ADX 10059, AZD-2066, AZD6538, AZD9272,basimglurant, dipraglurant, fenobam, GRN529, LY-344545, mavoglurant,MPEP, MRZ-8676, MTEP, rufinamide, SIB-1757, and SIB-1893.

In a particular embodiment, said combination of drugs used as analternative to acamprosate comprises a compound acting as an antagonistof both mGluR1 and mGluR5 receptors.

In an embodiment, combination of drugs used as an alternative toacamprosate comprises a drug that acts as an antagonist or negativeallosteric modulator of mGluR2 or mGluR3 (group II) metabotropicglutamate receptors.

In another embodiment, combination of drugs used as an alternative toacamprosate comprises a drug that acts as an antagonist or negativeallosteric modulator of mGluR2 and mGluR3 (group II) metabotropicglutamate receptors.

Antagonists of group 11 metabotropic glutamate receptors comprise, butare not limited to, EGLU, APTCA, LY-341495, BCI-632 or its prodrugsBCI-1038, BCI-1206, BCI-1283, BCI-838, or one of the 2-amino-bicyclo[3.1.0] hexane-2,6-dicarboxylic ester derivatives disclosed inWO2005000791, US2012028982, US2012004232, US2010298561, US2009306408.US2007021394, or 5H-thiazolo (3,2-a) pyrimidine derivatives disclosed inU.S. Pat. No. 5,958,931, which are incorporated herein by reference.

Negative allosteric modulators of the Group II metabotropic receptorsthat can be used in a combination of drugs used as an alternative toacamprosate comprise, but are not limited to, DT-2228 as disclosed inFroestl et al (2012) [53] and incorporated herein by reference, RG-1578,R04432717, RO4491533, dihydrobenzo [1,4] diazepin-2-one derivativesdisclosed in Hempstapat et al 2007 [54] and incorporated herein byreference, and compounds described in WO 2014/064028, WO 01/29011, WO01/29012, WO 02/083652, WO 02/083665, WO 02/098864, WO 03/066623, WO2005/014002, WO 2005/040171, WO2005/123738, WO 2006/084634, WO2006/099972, WO 2007/039439, WO 2007/110337 and WO 2008/119689 andincorporated herein by reference.

In another embodiment, said combination of drugs used as an alternativeto acamprosate comprises a drug that acts as an agonist or positiveallosteric modulator of the glycinic channels.

In a particular embodiment, said combination of drugs comprisesanandamide, cycloserine, dimethylglycine, dronabinol, enflurane,glycine, halothane, HU 210, hypotaurine, isoflurane, milacemide,sarcosine, serine, sevoflurane, taurine, trimethylglycine, D and/or Lalanine, or β alanine, which are known as agonists or positiveallosteric modulators of the glycinic channels.

Other agonists or positive allosteric modulators of the glycinicchannels that can be used in said combination of drugs comprise propofolderivatives described in WO 2010/067069, which are incorporated hereinby reference. Agonists or positive allosteric modulators of the glycinicchannels also comprise, but are not limited to, tropines and nortropineswhich are described in Maksay el al. (2007) [55] and are also hereinincorporated by reference, as well as those listed in Yevenes &Zeilhofer [56].

Therapy according to the invention may be provided at home, the doctor'soffice, a clinic, a hospital's outpatient department, or a hospital, sothat the doctor can observe the therapy's effects closely and make anyadjustments that are needed.

The duration of the therapy depends on the stage of the disease beingtreated, age and condition of the patient, and how the patient respondsto the treatment. The dosage, frequency and mode of administration ofeach component of the combination can be controlled independently. Forexample, one drug may be administered orally while the second drug maybe administered intramuscularly. Combination therapy may be given inon-and-off cycles that include rest periods so that the patient's bodyhas a chance to recover from any as yet unforeseen side effects. Thedrugs may also be formulated together such that one administrationdelivers all drugs.

The administration of each drug of the combination may be by anysuitable means that results in a concentration of the drug that,combined with the other component, is able to ameliorate the patient'scondition or efficiently treat the disease or disorder.

While it is possible for the drugs of the combination to be administeredas the pure chemicals, it is preferable to present them as apharmaceutical composition, also referred to in this context aspharmaceutical formulation. Possible compositions include those suitablefor oral, rectal, topical (including transdermal, buccal andsublingual), or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration.

More commonly these pharmaceutical formulations are prescribed to thepatient in “patient packs” containing a number of dosing units or othermeans for administration of metered unit doses for use during a distincttreatment period in a single package, usually a blister pack. Patientpacks have an advantage over traditional prescriptions, where apharmacist divides a patient's supply of a pharmaceutical from a bulksupply, in that the patient always has access to the package insertcontained in the patient pack, normally missing in traditionalprescriptions. The inclusion of a package insert has been shown toimprove patient compliance with the physician's instructions. Thus, theinvention further includes a pharmaceutical formulation, as hereinbefore described, in combination with packaging material suitable forsaid formulation. In such a patient pack the intended use of aformulation for the combination treatment can be inferred byinstructions, facilities, provisions, adaptations and/or other means tohelp use the formulation most suitably for the treatment. Such measuresmake a patient pack specifically suitable for and adapted for use fortreatment with the combination of the present invention.

The drug may be contained, in any appropriate amount, in any suitablecarrier substance. The drug may be present in an amount of up to 99% byweight of the total weight of the composition. The composition may beprovided in a dosage form that is suitable for the oral, parenteral(e.g., intravenous, intramuscular), rectal, cutaneous, nasal, vaginal,inhalant, skin (patch), or ocular administration route. Thus, thecomposition may be in the form of, e.g., tablets, capsules, pills,powders, granulates, suspensions, emulsions, solutions, gels includinghydrogels, pastes, ointments, creams, plasters, drenches, osmoticdelivery devices, suppositories, enemas, injectables, implants, sprays,or aerosols.

The pharmaceutical compositions may be formulated according toconventional pharmaceutical practice (see, e.g., Remington: The Scienceand Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, LippincottWilliams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology,eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

Pharmaceutical compositions according to the invention may be formulatedto release the active drug substantially immediately upon administrationor at any predetermined time or time period after administration.

The controlled release formulations include (i) formulations that createa substantially constant concentration of the drug within the body overan extended period of time; (ii) formulations that after a predeterminedlag time create a substantially constant concentration of the drugwithin the body over an extended period of time, (iii) formulations thatsustain drug action during a predetermined time period by maintaining arelatively constant, effective drug level in the body with concomitantminimization of undesirable side effects associated with fluctuations inthe plasma level of the active drug substance; (iv) formulations thatlocalize drug action by, e.g., spatial placement of a controlled releasecomposition adjacent to or in the diseased tissue or organ; and (v)formulations that target drug action by using carriers or chemicalderivatives to deliver the drug to a particular target cell type.

Administration of drugs in the form of a controlled release formulationis especially preferred in cases in which the drug has (i) a narrowtherapeutic index (i.e., the difference between the plasma concentrationleading to harmful side effects or toxic reactions and the plasmaconcentration leading to a therapeutic effect is small; in general, thetherapeutic index, TI, is defined as the ratio of median lethal dose(LD50) to median effective dose (ED50)); (ii) a narrow absorption windowin the gastrointestinal tract; or (iii) a very short biologicalhalf-life so that frequent dosing during a day is required in order tosustain the plasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtaincontrolled release in which the rate of release outweighs the rate ofmetabolism of the drug in question. Controlled release may be obtainedby appropriate selection of various formulation parameters andingredients, including, e.g., various types of controlled releasecompositions and coatings. Thus, the drug is formulated with appropriateexcipients into a pharmaceutical composition that, upon administration,releases the drug in a controlled manner (single or multiple unit tabletor capsule compositions, oil solutions, suspensions, emulsions,microcapsules, microspheres, nanoparticles, patches, and liposomes).

Solid Dosage Forms for Oral Use Formulations for oral use includetablets containing the composition of the invention in a mixture withnon-toxic pharmaceutically acceptable excipients. These excipients maybe, for example, inert diluents or fillers (e.g., sucrose,microcrystalline cellulose, starches including potato starch, calciumcarbonate, sodium chloride, calcium phosphate, calcium sulfate, orsodium phosphate); granulating and disintegrating agents (e.g.,cellulose derivatives including microcrystalline cellulose, starchesincluding potato starch, croscarmellose sodium, alginates, or alginicacid); binding agents (e.g., acacia, alginic acid, sodium alginate,gelatin, starch, pregelatinized starch, microcrystalline cellulose,carboxymethylcellulose sodium, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethyleneglycol); or lubricating agents, glidants, or antiadhesives (e.g.,stearic acid, silicas, or talc). Other pharmaceutically acceptableexcipients can be colorants, flavoring agents, plasticizers, humectants,buffering agents, and the like.

The tablets may be uncoated or they may be coated by known techniques,optionally to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. The coating may be adapted to release the active drugsubstance in a predetermined pattern (e.g., in order to achieve acontrolled release formulation) or it may be adapted not to release theactive drug substance until after passage of the stomach (entericcoating). The coating may be a sugar coating, a film coating (e.g.,based on hydroxypropyl methylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose,acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone),or an enteric coating (e.g., based on methacrylic acid copolymer,cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate,hydroxypropyl methylcellulose acetate succinate, polyvinyl acetatephthalate, shellac, and/or ethylcellulose). A time delay material suchas, e.g., glyceryl monostearate or glyceryl distearate may be employed.

The solid tablet compositions may include a coating adapted to protectthe composition from unwanted chemical changes, (e.g., chemicaldegradation prior to the release of the active drug substance). Thecoating may be applied on the solid dosage form in a similar manner tothat described in Encyclopedia of Pharmaceutical Technology.

Drugs may be mixed together in the tablet, or may be partitioned. Forexample, a first drug is contained on the inside of the tablet, and asecond drug is on the outside, such that a substantial portion of thesecond drug is released prior to the release of the first drug.

Formulations for oral use may also be presented as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent (e.g., potato starch, microcrystalline cellulose,calcium carbonate, calcium phosphate or kaolin), or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium, for example, liquid paraffin or olive oil. Powders andgranulates may be prepared using the ingredients mentioned above undertablets and capsules in a conventional manner.

Controlled release compositions for oral use may, e.g., be constructedto release the active drug by controlling the dissolution and/or thediffusion of the active drug substance.

Dissolution or diffusion-controlled release can be achieved byappropriate coating of a tablet, capsule, pellet, or granulatedformulation of drugs, or by incorporating the drug into an appropriatematrix. A controlled release coating may include one or more of thecoating substances mentioned above and/or, e.g., shellac, beeswax,glycowax, castor wax, carnauba wax, stearyl alcohol, glycerylmonostearate, glyceryl distearate, glycerol palmitostearate,ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetatebutyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone,polyethylene, polymethacrylate, methylmethacrylate,2-hydroxymethacrylate, methacrylate hydrogel, 1,3 butylene glycol,ethylene glycol methacrylate, and/or polyethylene glycol. In acontrolled release matrix formulation, the matrix material may alsoinclude, e.g., hydrated methylcellulose, carnauba wax and stearylalcohol, carbopol 934, silicone, glyceryl tristearate, methylacrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/orhalogenated fluorocarbon.

A controlled release composition containing one or more of the drugs ofthe claimed combinations may also be in the form of a buoyant tablet orcapsule (i.e., a tablet or capsule that, upon oral administration,floats on top of the gastric content for a certain period of time). Abuoyant tablet formulation of the drug(s) can be prepared by granulatinga mixture of the drug(s) with excipients and 20-75% w/w ofhydrocolloids, such as hydroxyethylcellulose, hydroxypropylcellulose, orhydroxypropylmethylcellulose. The obtained granules can then becompressed into tablets. On contact with the gastric juice, the tabletforms a substantially water-impermeable gel barrier around its surface.This gel barrier takes part in maintaining a density of less than one,thereby allowing the tablet to remain buoyant in the gastric juice.

Liquids for Oral Administration

Powders, dispersible powders, or granules suitable for preparation of anaqueous suspension by addition of water are convenient dosage forms fororal administration. Formulation as a suspension provides the activeingredient in a mixture with a dispersing or wetting agent, suspendingagent, and one or more preservatives. Suitable suspending agents are,for example, sodium carboxymethylcellulose, methylcellulose, sodiumalginate, and the like.

Parenteral Compositions

The pharmaceutical composition may also be administered parenterally byinjection, infusion or implantation (intravenous, intramuscular,subcutaneous, or the like) in dosage forms, formulations, or viasuitable delivery devices or implants containing conventional, non-toxicpharmaceutically acceptable carriers and adjuvants. The formulation andpreparation of such compositions are well-known to those skilled in theart of pharmaceutical formulation.

Compositions for parenteral use may be provided in unit dosage forms(e.g., in single-dose ampoules), or in vials containing several dosesand in which a suitable preservative may be added (see below). Thecomposition may be in form of a solution, a suspension, an emulsion, aninfusion device, or a delivery device for implantation or it may bepresented as a dry powder to be reconstituted with water or anothersuitable vehicle before use. Apart from the active drug(s), thecomposition may include suitable parenterally acceptable carriers and/orexcipients. The active drug(s) may be incorporated into microspheres,microcapsules, nanoparticles, liposomes, or the like for controlledrelease. The composition may include suspending, solubilizing,stabilizing, pH-adjusting, and/or dispersing agents.

The pharmaceutical compositions according to the invention may be in aform suitable for sterile injection. To prepare such a composition, thesuitable active drug(s) are dissolved or suspended in a parenterallyacceptable liquid vehicle. Among acceptable vehicles and solvents thatmay be employed are water, water adjusted to a suitable pH by additionof an appropriate amount of hydrochloric acid, sodium hydroxide or asuitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodiumchloride solution. The aqueous formulation may also contain one or morepreservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). Incases where one of the drugs is only sparingly or slightly soluble inwater, a dissolution enhancing or solubilizing agent can be added, orthe solvent may include 10-60% w/w of propylene glycol or the like.

Controlled release parenteral compositions may be in form of aqueoussuspensions, microspheres, microcapsules, magnetic microspheres, oilsolutions, oil suspensions, or emulsions. Alternatively, the activedrug(s) may be incorporated in biocompatible carriers, liposomes,nanoparticles, implants, or infusion devices. Materials for use in thepreparation of microspheres and/or microcapsules are, e.g.,biodegradable/bioerodible polymers such as polygalactin, poly-(isobutylcyanoacrylate), and poly(2-hydroxyethyl-L-glutamine). Biocompatiblecarriers that may be used when formulating a controlled releaseparenteral formulation are carbohydrates (e.g., dextrans), proteins(e.g., albumin), lipoproteins, or antibodies. Materials for use inimplants can be non-biodegradable (e.g., polydimethyl siloxane) orbiodegradable (e.g., poly(caprolactone), poly(glycolic acid) orpoly(ortho esters)).

Alternative Routes

Although less preferred and less convenient, other administrationroutes, and therefore other formulations, may be contemplated. In thisregard, for rectal application, suitable dosage forms for a compositioninclude suppositories (emulsion or suspension type) and rectal gelatincapsules (solutions or suspensions). In a typical suppositoryformulation, the active drug(s) are combined with an appropriatepharmaceutically acceptable suppository base such as cocoa butter,esterified fatty acids, glycerinated gelatin, and various water-solubleor dispersible bases like polyethylene glycol. Various additives,enhancers, or surfactants may be incorporated.

The pharmaceutical compositions may also be administered topically onthe skin for percutaneous absorption in dosage forms or formulationscontaining conventionally non-toxic pharmaceutical acceptable carriersand excipients including microspheres and liposomes. The formulationsinclude creams, ointments, lotions, liniments, gels, hydrogels,solutions, suspensions, sticks, sprays, pastes, plasters, and otherkinds of transdermal drug delivery systems. The pharmaceuticallyacceptable carriers or excipients may include emulsifying agents,antioxidants, buffering agents, preservatives, humectants, penetrationenhancers, chelating agents, gel-forming agents, ointment bases,perfumes, and skin protective agents.

The preservatives, humectants, and penetration enhancers may beparabens, such as methyl or propyl p-hydroxybenzoate, benzalkoniumchloride, glycerin, propylene glycol, urea, etc.

The pharmaceutical compositions described above for topicaladministration on the skin may also be used in connection with topicaladministration onto or close to the part of the body that is to betreated. The compositions may be adapted for direct application or forapplication by means of special drug delivery devices such as dressingsor, alternatively, plasters, pads, sponges, strips, or other forms ofsuitable flexible material.

Dosages and Duration of the Treatment

It will be appreciated that the drugs of the combination may beadministered concomitantly, either in the same or differentpharmaceutical formulation or sequentially. If there is sequentialadministration, the delay in administering the second (or additional)active ingredient should not be such as to lose the benefit of theefficacious effect of the combination of the active ingredients. Aminimum requirement for a combination according to this description isthat the combination should be intended for combined use with thebenefit of the efficacious effect of the combination of the activeingredients. The intended use of a combination can be inferred byfacilities, provisions, adaptations and/or other means to help use thecombination according to the invention.

Therapeutically effective amounts of the drugs in a combination of thisinvention include, e.g., amounts that are effective for reducingAlzheimer's disease symptoms, halting or slowing the progression of thedisease once it has become clinically manifest, or preventing orreducing the risk of developing the disease.

Although the active drugs of the present invention may be administeredin divided doses, for example two or three times daily, a single dailydose of each drug in the combination is preferred, with a single dailydose of all drugs in a single pharmaceutical composition (unit dosageform) being most preferred.

Administration can be one to several times daily for several days toseveral years, and may even be for the life of the patient. Chronic orat least periodically repeated long-term administration is indicated inmost cases.

The term “unit dosage form” refers to physically discrete units (such ascapsules, tablets, or loaded syringe cylinders) suitable as unitarydosages for human subjects, each unit containing a predeterminedquantity of active material or materials calculated to produce thedesired therapeutic effect, in association with the requiredpharmaceutical carrier.

The amount of each drug in a preferred unit dosage composition dependsupon several factors including the administration method, the bodyweight and the age of the patient, the stage of the disease, and therisk of potential side effects considering the general health status ofthe person to be treated. Additionally, pharmacogenomic (the effect ofgenotype on the pharmacokinetic, pharmacodynamic or efficacy profile ofa therapeutic) information about a particular patient may affect thedosage used.

Except when responding to especially impairing cases, where higherdosages may be required, the preferred dosage of each drug in thecombination will usually lie within the range of doses not above thedosage usually prescribed for long-term maintenance treatment or provento be safe in phase 3 clinical studies.

One remarkable advantage of the invention is that each compound may beused at low doses in a combination therapy, while producing, incombination, a substantial clinical benefit to the patient. Thecombination therapy may indeed be effective at doses where the compoundsindividually have little or no effect. Accordingly, a particularadvantage of the invention lies in the ability to use sub-optimal dosesof each compound, i.e., doses which are lower than therapeutic dosesusually prescribed, preferably ½ of therapeutic doses, more preferably⅓, ¼, ⅕, or even more preferably 1/10 of therapeutic doses. Inparticular examples, doses as low as 1/20, 1/30, 1/50, 1/100, or evenlower, of therapeutic doses are used.

At such sub-therapeutic dosages, the compounds would exhibit no sideeffects, while the combination(s) according to the invention are fullyeffective in treating Alzheimer's disease or AD related diseases.

A preferred dosage corresponds to amounts from 1% up to 50% of thoseusually prescribed for long-term maintenance treatment.

The most preferred dosage may correspond to amounts from 1% up to 10% ofthose usually prescribed for long-term maintenance treatment.

Specific examples of dosages of drugs for use in the invention areprovided below:

-   -   Acamprosate between 0.1 and 1000 mg per day, preferably less        than 400 mg per day, more preferably less than 200 mg per day,        even more preferably 100 mg per day or less, furthermore        preferably between 0.5 mg and 100 mg per day, typically 0.8 mg        per day, 2 mg per day, 20 mg per day, 40 mg per day, or 80 mg        per day, such dosages being particularly suitable for oral        administration,    -   Baclofen between 0.01 to 150 mg per day, preferably less than        100 mg per day, more preferably less than 50 mg per day, most        preferably between 5 and 40 mg per day, even more preferably        less than 35 mg per day, typically 12 mg per day, 24 mg per day,        or 30 mg per day, such dosages being particularly suitable for        oral administration,    -   Aminocaproic acid orally from about 0.1 g to 2.4 g per day,    -   bromocriptine orally from about 0.01 to 10 mg per day,    -   diethylcarbamazine orally from about 0.03 to 400 mg per day,    -   cabergoline orally from about 1 to 10 μg per day,    -   cinacalcet orally from about 0.3 to 36 mg per day,    -   cinnarizine orally from about 0.6 to 23 mg per day,    -   dyphylline orally from about 9 to 320 mg per day,    -   eplerenone orally from about 0.25 to 10 mg per day,    -   ifenprodil orally from about 0.4 to 6 mg per day,    -   leflunomide orally from about 0.1 to 10 mg per day,    -   levosimendan orally from about 0.04 to 0.8 mg per day,    -   mexiletine orally from about 6 to 120 mg per day,    -   moxifloxacin orally from about 4 to 40 mg per day,    -   phenformin orally from about 0.25 to 15 mg per day,    -   quinacrine orally from about 1 to 30 mg per day,    -   sulfisoxazole orally from about 20 to 800 mg per day,    -   sulodexide orally from about 0.05 to 40 mg per day,    -   terbinafine orally from about 2.5 to 25 mg per day,    -   torasemide orally from about 0.05 to 4 mg per day,    -   trimetazidine orally from about 0.4 to 6 mg per day, and    -   zonisamide orally from about 0.5 to 50 mg per day, administered        in one, two, or three doses daily.

When the composition comprises, as active ingredients, only baclofen andacamprosate, these two compounds may be used in different ratios, e.g.,at a weight ratio of acamprosate/baclofen comprised from between 0.05 to1000 (W:W), preferably between 0.05 to 100 (W:W), more preferablybetween 0.05 to 50 (W:W).

As already mentioned, the above compounds in the therapies of theinvention can be used as specifically designated per se, as well as anypharmaceutically acceptable salts, enantiomers, racemates, prodrugs,metabolites or derivatives thereof.

In a particularly preferred embodiment, combinatorial therapies of theinvention comprise administering between 0.4 mg and 50 mg acamprosateand 6 mg to 15 mg baclofen, twice daily.

In an embodiment, combinatorial therapies of the invention compriseadministering 0.4 mg acamprosate and 6 mg baclofen, twice daily.

In a preferred embodiment, combinatorial therapies of the inventioncomprise administering 1 mg acamprosate and 15 mg baclofen, twice daily.

In yet another preferred embodiment, combinatorial therapies of theinvention comprise administering 10 mg acamprosate and 6 mg baclofen,twice daily.

In another preferred embodiment, combinatorial therapies of theinvention comprise administering 20 mg acamprosate and 12 mg baclofen,twice daily.

In still another embodiment, combinatorial therapies of the inventioncomprise administering 40 mg acamprosate and 12 mg baclofen, twicedaily.

In another particular embodiment, besides comprising administering oneof the above baclofen-acamprosate regimens, therapies of the inventionalso comprise administering donepezil or memantine either at their usualdose and regimen (i.e., as an add-on therapy) or even at a lower dose,from 1% up to 50% of those usually prescribed for the treatment of AD.

In an even more particular embodiment, combinatorial therapies of theinvention further comprise administering 0.6 mg donepezil twice daily.

Consequently, in a particularly preferred embodiment, combinatorialtherapies of the invention comprise administering between 0.4 mg and 50mg of acamprosate, 6 mg to 15 mg baclofen, and 0.6 mg donepezil, twicedaily.

In an embodiment, combinatorial therapies the invention compriseadministering 0.4 mg acamprosate, 6 mg baclofen, and 0.6 mg donepezil,twice daily.

In a preferred embodiment, combinatorial therapies of the inventioncomprise administering 1 mg acamprosate, 15 mg baclofen, and 0.6 mgdonepezil, twice daily.

In yet another preferred embodiment, combinatorial therapies of theinvention comprise administering 20 mg acamprosate, 12 mg baclofen, and0.6 mg donepezil, twice daily.

In a particularly preferred embodiment, combinatorial therapies of theinvention comprise administering 40 mg acamprosate, 12 mg baclofen, and0.6 mg donepezil, twice daily.

As previously stated, the compounds in a combinatorial therapy of theinvention may be administered simultaneously, separately, sequentiallyand/or repeatedly to the subject. Particularly, the above dosingregimens can be simultaneously orally administered using suitabletablets. Also, a particular object of this invention relates to a tabletcomprising acamprosate and baclofen, suitable for the dosing regimen ofa dose of acamprosate between 0.4 and 50 mg and a dose of baclofenbetween 6 and 15 mg, to be administered twice daily.

A further particular object of this invention relates to a tabletcomprising acamprosate and baclofen, suitable for the dosing regimen of0.4 mg acamprosate and 6 mg baclofen, twice daily.

Another particular object of this invention relates to a tabletcomprising acamprosate and baclofen, suitable for the dosing regimen of1 mg acamprosate and 15 mg baclofen, twice daily.

Another particular object of this invention relates to a tabletcomprising acamprosate and baclofen, suitable for the dosing regimen of20 mg acamprosate and 12 mg baclofen, to be administered twice daily.

A particular object of this invention relates to a tablet comprisingacamprosate and baclofen, suitable for the dosing regimen of 40 mgacamprosate and 12 mg baclofen, to be administered twice daily.

An even more particular object of this invention relates to a tabletcomprising baclofen, acamprosate and donepezil, said tablet beingsuitable for any of the above mentioned dosages of baclofen andacamprosate and also for the dosing regimen of 0.6 mg donepezil, twicedaily.

A more particular object of the invention is a scored tablet suitablefor the administration of any of the above dosing regimens, said tabletbeing cleavable in 2, 3 and/or 4 part as a function of the dose to beadministered at each taking.

As mentioned above, a single unit dosage form containing thecombinations of the invention is most preferred. Alternatively, where aseparate administration would be considered more proper, combinations ofthe invention can be provided in the form of a unit dosage package, suchunit dosage package being configured to hold a first unit dosagecomprising acamprosate and a second unit dosage comprising baclofen. Ina particular embodiment unit dosages are tablets. In another particularembodiment the first unit dosage comprises between 0.4 and 50 mg ofacamprosate and the second unit dosage comprises between 6 and 15 mgbaclofen.

In another embodiment the first unit dosage is suitable for the dosingregimen of 0.4 mg acamprosate, twice daily.

In another embodiment the first unit dosage is suitable for the dosingregimen of 1 mg acamprosate, twice daily.

In another embodiment the first unit dosage is suitable for the dosingregimen of 20 mg acamprosate, twice daily.

In another embodiment the first unit dosage is suitable for the dosingregimen of 40 mg acamprosate, twice daily.

In an embodiment the first unit dosage is suitable for the dosingregimen of 1 mg acamprosate, twice daily.

In another embodiment the second unit dosage is suitable for the dosingregimen of 6 mg baclofen, twice daily.

In another embodiment the second unit dosage is suitable for the dosingregimen of 12 mg baclofen, twice daily.

In another embodiment the second unit dosage is suitable for the dosingregimen of 15 mg baclofen, twice daily.

In another particular embodiment, the above mentioned unit dosagepackage comprises a third unit dosage comprising donepezil. In a moreparticular embodiment this third unit dosage is suitable for the dosingregimen of 0.6 mg donepezil, twice daily. In a particular embodimentsaid unit dosage package contains a number of unit dosages suitable for1, 2, 3, 4, 5, 6, 7 days of treatment or even more, preferably severalweeks of treatment.

It will be understood that the amount of the drug actually administeredwill be determined by a physician, in light of the relevantcircumstances including the condition to be treated, the exactcomposition to be administered, the age, weight, and response of theindividual patient, the severity of the patient's symptoms, and thechosen route of administration. Therefore, the above dosage ranges areintended to provide general guidance and support for the teachingsherein, but are not intended to limit the scope of the invention.

The following examples are given for purposes of illustration and not byway of limitation.

Examples

The care and husbandry of animals as well as the experiments areperformed according to the guidelines of the Committee for Research andEthical Issue of the I.A.S.P. (1983).

I. Treatment of Diseases Related to Aβ Toxicity

In this series of experiments, candidate combinations have been testedfor their ability to prevent or reduce the toxic effects of humanAβ₁₋₄₂. Aβ₁₋₄₂ is the full-length peptide that constitutes aggregatesfound in biopsies from human patients afflicted with AD. The effect isdetermined on various cell types, to further document the activity ofthe combinations in in vitro models which illustrate differentphysiological features of AD. In vivo studies are also performed in amouse model for AD, confirming this protective effect by evaluating theeffect of the combinations on i) the cognitive performance of theanimals and ii) molecular hallmarks (apoptosis induction, oxidativestress induction, inflammation pathway induction) of AD. Clinicalresults show that baclofen and acamprosate-based compositions areactually efficient in improving cognitive performance, as well as incorrecting electrophysiological disturbances observed in patientsdiagnosed with mild AD.

A. Baclofen-Acamprosate Combination Therapies Prevent Toxicity of HumanAβ₁₋₄₂ In Vitro

1. Effect on the Toxicity of Human Aβ₁₋₄₂ Peptide on Human BrainMicrovascular Endothelial Cells (HBMEC).

HBMEC Culture Conditions

HBMEC cultures were used to study the protection afforded by candidatecompound(s) on Aβ₁₋₄₂ toxicity.

HBMEC (ScienCell Ref: 1000, frozen at passage 10) were rapidly thawed ina water bath at +37° C. The supernatant was immediately put in 9 mlDulbecco's modified Eagle's medium (DMEM; Pan Biotech ref: P04-03600)containing 10% fetal calf serum (FCS; GIBCO ref 10270-106). Cellsuspension was centrifuged at 180×g for 10 min at +4° C. and the pelletswere suspended in CSC serum-free medium (CSC serum-free medium. CellSystem, Ref: SF-4Z0-500-R, Batch 51407-4) with 1.6% serum-freeRocketFuel (Cell System, Ref: SF-4Z0-500-R, Batch 54102), 2% ofPenicillin 10,000 U/ml and Streptomycin 10 mg/ml (PS; Pan Biotech ref:P06-07100 batch 133080808) and were seeded at the density of 20,000cells per well in 96-well plates (matrigel layer biocoat angiogenesissystem, BD, Ref 354150, Batch A8662) in a final volume of 100 μl. Onmatrigel support, endothelial cerebral cells spontaneously started theprocess of capillary network morphogenesis [57].

Three separate cultures were performed per condition, 6 wells percondition.

Test Compounds and Human Aβ₁₋₄₂ Treatment

Briefly, Aβ₁₋₄₂ peptide (Bachem, ref: H1368 batch 1010533) wasreconstituted in defined culture medium at 20 μM (mother solution) andwas slowly shaken at +37° C. for 3 days in the dark. The control mediumwas prepared in the same conditions.

After 3 days, human amyloid peptide was used on HBMEC at 2.5 μM dilutedin control medium (optimal incubation time). The Aβ₁₋₄₂ peptide wasadded 2 hours after HBMEC seeding on matrigel for 18 hours incubation.

One hour after HBMEC seeding on matrigel, test compounds and VEGF-165were solved in culture medium (+0.1% DMSO) and then pre-incubated withHBMEC for 1 hour before the Aβ₁₋₄₂ application (in a final volume perculture well of 100 μl). One hour after test compounds or VEGFincubation (two hours after cell seeding on matrigel), 1001l of Aβ₁₋₄₂peptide was added to a final concentration of 2.5 μM diluted in controlmedium in the presence of test compounds or VEGF (in a 200 μl totalvolume/well), in order to avoid further drug dilutions

Organization of Culture Plates

VEGF-165, known to be a pro-angiogenic isoform of VEGF-A, was used forall experiments in this study as reference compound. VEGF-165 is one ofthe most abundant VEGF isoforms involved in angiogenesis. VEGF was usedas reference test compound at 10 nM (FIG. 1).

The following conditions were assessed:

-   -   Negative Control: medium alone+0.1% DMSO.    -   Intoxication: amyloid-β₁₋₄₂ (2.5 μM) for 18 h.    -   Positive control: VEGF-165 (10 nM) (1 reference        compound/culture) 1 h before the Aβ₁₋₄₂ (2.5 μM) addition for an        18 h incubation time.    -   Test compounds: Test compound(s) 1 h before the Aβ₁₋₄₂ (2.5 μM)        addition for an 18 h incubation time.

Capillary Network Quantification

Per well, 2 pictures with 4× lens were taken using InCell Analyzer™ 1000(GE Healthcare) in light transmission. All images were taken in the sameconditions. Analysis of the angiogenesis networks was done usingDeveloper software (GE Healthcare). The total length of capillarynetwork was assessed.

Data Processing

Data were expressed in percentage of control conditions (nointoxication, no amyloid=100%) in order to express the amyloid injury.All values were expressed as mean+/−SEM (s.e. mean) of the 3 cultures(n=6 wells per condition). Statistical analyses were done on thedifferent conditions (ONE-WAY ANOVA followed by the Dunnett's test whenit was allowed, Statview software version 5.0).

Results

The baclofen-acamprosate combination gives a significant protectiveeffect against toxicity of human Aβ₁₋₄₂ peptide in an HBMEC model (areduction of 24% of Aβ₁₋₄₂ peptide injury is observed), as shown in FIG.2. The results clearly show that the intoxication by human amyloidpeptide (Aβ₁₋₄₂ 2.5 μM) is significantly prevented by the drugcombination whereas, at those concentrations, the drugs alone have nosignificant effect on intoxication in the experimental conditionsdescribed above.

Conversely, the combination of baclofen and terbinafine (which ispresented here only for the sake of comparison) affords a weakerprotection (a reduction of 15% of Aβ₁₋₄₂ peptide injury is observed)against Aβ₁₋₄₂ (FIG. 3).

Thus, although these two combinations allow a protection against Aβ₁₋₄₂,the combination of baclofen-acamprosate clearly stands out. Indeed,these drugs at concentrations having no effect alone allow significantprotection of human HBMEC against Aβ₁₋₄₂ when used in combination.Furthermore, the baclofen-acamprosate combination is more effective thanthe baclofen-terbinafine combination. Such an effect of baclofen andacamprosate represents a remarkable improvement, by 60%, in comparisonto, e.g., the effect of the combination of baclofen-terbinafine.

Moreover, the concentration of baclofen used in the baclofen-acamprosatecombination is much lower than the concentration of baclofen used in thebaclofen-terbinafine combination (25-fold reduction).

2. Effect on the Toxicity of Human a Aβ₁₋₄₂ Peptide on Primary CorticalNeuron Cells.

a) Baclofen-Acamprosate Combination has a Neuroprotective Effect

Culture of Primary Cortical Neurons

Rat cortical neurons were cultured as described by Singer et al. [58].Briefly pregnant female rats of 15 days gestation were killed bycervical dislocation (Rats: Wistar) and the foetuses were removed fromthe uterus. The cortex was removed and placed in ice-cold medium ofLeibovitz (L15) containing 2% of Penicillin 10,000 U/ml and Streptomycin10 mg/ml and 1% of bovine serum albumin (BSA). Cortices were dissociatedby trypsin for 20 min at 37° C. (0.05%). The reaction was stopped by theaddition of Dulbecco's modified Eagle's medium (DMEM) containing DNaseIgrade II and 10% of foetal calf serum (FCS). Cells were thenmechanically dissociated by 3 serial passages through a 10 ml pipetteand centrifuged at 515×g for 10 min at +4° C. The supernatant wasdiscarded and the pellet of cells was re-suspended in a defined culturemedium consisting of Neurobasal supplemented with B27 (2%), L-glutamine(0.2 mM), 2% of PS solution and 10 ng/ml of BDNF. Viable cells werecounted in a Neubauer cytometer using the trypan blue exclusion test.The cells were seeded at a density of 30,000 cells/well in 96-wellplates (wells were pre-coated with poly-L-lysine (10 μg/ml)) and werecultured at +37° C. in a humidified air (95%)/CO₂ (5%) atmosphere.

Three independent cultures will be performed per condition, 6 wells percondition.

Test Compounds and Human Amyloid-β₁₋₄₂ Treatment

Briefly, Aβ₁₋₄₂ peptide was reconstituted in defined culture medium at40 μM (mother solution) and was slowly shaken at +37° C. for 3 days inthe dark. The control medium was prepared in the same conditions.

After 3 days, the solution was used on primary cortical neurons asfollows.

After 10 days of neuron culture, test compounds were solved in culturemedium (+0.1% DMSO) and then pre-incubated with neurons for one hourbefore the Aβ₁₋₄₂ application (in a final volume per culture well of 100μl). One hour after test compound(s) incubation, 100 μl of Aβ₁₋₄₂peptide was added to a final concentration of 10 μM diluted in thepresence of the drug(s), in order to avoid further test compound(s)dilutions. Cortical neurons were intoxicated for 24 h. Three separatecultures were performed per condition, 6 wells per condition.

BDNF (50 ng/ml) and Estradiol-β (150 nM) were used as positive controland reference compounds respectively. Three separate cultures wereperformed per condition, 12 wells per condition.

Organization of Cultures Plates

Estradiol-β at 150 nM was used as a positive control (FIG. 4).

Estradiol-β was solved in culture medium and pre-incubated for 1 hbefore the amyloid-β₁₋₄₂ application.

The following conditions were assessed:

-   -   CONTROL PLAQUE: 12 wells/condition        -   Negative Control: medium alone+0.1% DMSO,        -   Intoxication: amyloid-β₁₋₄₂ (10 μM) for 24 h, and        -   Reference compound: Estradiol (150 nM) t h.    -   DRUG PLATE: 6 wells/condition    -   Negative Control: medium alone+0.1% DMSO,    -   Intoxication: amyloid-β₁₋₄₂ (10 μM) for 24 h, and    -   Test compound(s): test compound(s)—1 h followed by amyloid-β₁₋₄₂        (10 μM) for 24 h.

Lactate Dehydrogenase (LDH) Activity Assay

24 hours after intoxication, the supernatant was taken off and analyzedwith the Cytotoxicity Detection Kit (LDH, Roche Applied Science, ref:11644793001, batch: 11800300). This colorimetric assay for thequantification of cell toxicity is based on the measurement of lactatedehydrogenase activity released from the cytosol of dying cells into thesupernatant.

Data Processing

Data were expressed in percentage of control conditions (nointoxication, no amyloid=100%) in order to express the amyloid injury.All values were expressed as mean+/−SEM (s.e. mean) of the 3 cultures(n=6 wells per condition). Statistical analyses were done on thedifferent conditions (one-way ANOVA followed by the Dunnett's test whenit was allowed, Statview software version 5.0).

Results

The combination of baclofen and acamprosate induces a significantprotective effect against the toxicity of human Aβ₁₋₄₂ peptide(improvement of 34% of cell survival) in primary cortical neuron cells,as shown in FIG. 5. The results clearly show that intoxication by humanamyloid peptide (Aβ₁₋₄₂ 10 μM) is significantly prevented by thecombination, whereas at those concentrations, baclofen or acamprosatealone have no significant effect on intoxication.

Conversely, although active in this model, the combination ofsulfisoxazole and cinacalcet affords a weaker protection against Aβ₁₋₄₂(19%, FIG. 6).

Thus, while those two combinations allow a protection against Aβ₁₋₄₂,the combination baclofen-acamprosate stands out clearly. Indeed, atconcentrations having no effect alone, the drugs cause a significantprotection of primary cortical neuron cells against Aβ₁₋₄₂ when used incombination. Furthermore, the baclofen-acamprosate combination is muchmore effective than the sulfisoxazole-cinacalcet combination Such aneffect of baclofen and acamprosate represents a remarkable improvementby 60% in comparison to, e.g., the effect of the combination ofsulfisoxazole and cinacalcet.

Taken together these results show an unexpected and remarkable positiveeffect of baclofen-acamprosate combinations in several in vitro modelsof Alzheimer's disease. The effect observed is highly superior to thatprovoked by other baclofen-based combination therapies (e.g.,baclofen-terbinafine), or other active combination therapies(sulfisoxazole-cinacalcet).

A comparison of acamprosate and homotaurine protection activity oncortical cells has been done (FIG. 17). Those results show that thederivative of acamprosate, called homotaurine, allows an effectiveprotection against Aβ₁₋₄₂. In the context of this invention, baclofen oracamprosate can thus be substituted by their derivatives, provided thatthose derivatives are efficient in the assay described herein.

b) Cellular Pathways Triggered in Neurons by Aβ₁₋₄₂ Oligomers areReversed Upon Baclofen-Acamprosate Treatment

Mitochondrial dysfunction is thought to play a critical role in AD byproducing two major cellular consequences: oxidative stress and celldeath by apoptosis. The effect of baclofen-acamprosate treatment onoxidative stress and apoptosis generated by Aβ oligomers has beenassessed by measuring the oxidation of methionine residues (methioninesulfoxide, MetO) and by measuring the release of mitochondrialcytochrome C (Cyto C, as a marker of early apoptosis) into the cytoplasmof intoxicated neuronal cells. The effect on Aβ-induced apoptosis wasfurther confirmed by measuring the level of caspase 3, a marker of lateapoptosis.

Other hallmarks of AD are i) the accumulation of hyperphosphorylated Tauproteins (pTau) resulting in the formation of neurofibrillary tanglesand ii) excitotoxicity due to an excess of glutamate. The effect ofbaclofen-acamprosate treatment on Tau phosphorylation withinAβ-intoxicated neuronal cells and on glutamate accumulation in culturemedium was assessed (FIG. 21).

Culture of Primary Cortical Neurons, Aβ₁₋₄₂ Oligomer Formation and CellIntoxication

Cell culture, formation of Aβ₁₋₄₂ oligomer and cell intoxication weremainly performed as above. Variable intoxication times and Aβconcentrations were used depending on the experiment and are summarizedin Table 5 below.

TABLE 5 Aβ₁₋₄₂ Days Seeding Intoxication of density time Assay culture(cells/well) Concentration (h) MetO 11 15,000 1.25 μM 4 Cyto c 11 30,0001.25 μM 4 Caspase 3 11 30,000   10 μM 24 Tau 11 30,000  2.5 μM 16phosphorylation Glutamate release 13 30,000  2.5 μM 4Methionine Sulfoxide (MetO), Cytochrome c (Cyto c), Caspase 3, andPhosphorylated Tau (pTau) Assays:

After Aβ₁₋₄₂ intoxication, cells were fixed, permeabilized, andnon-specific sites were blocked with a solution of phosphate bufferedsaline (PBS; PanBiotech) containing 0.1% of saponin (Sigma) and 1% FCS.Then, cells were incubated with one of the assay specific primaryantibodies (Rabbit polyclonal anti MetO, 1/100, Euromedex, France;Rabbit polyclonal anti Cyto c, 1/100, Abcam; Rabbit polyclonal antiCaspase 3, 1/500, Sigma; Mouse monoclonal anti PHF-Tau, clone AT100,1/100, Thermo Scientific) and with MAP2 primary antibody (Mousemonoclonal anti MAP2, 1/400, Sigma Aldrich or Chicken polyclonal antiMAP2, 1/400, Abcam), which are revealed with the fitting secondaryantibodies (Alexa Fluor 488 goat anti mouse IgG; Alexa Fluor 568 goatanti rabbit IgG; Alexa Fluor 568 goat anti chicken IgG, all the threefrom Invitrogen and used at the 1/400 dilution).

Nuclei were counter-stained with Hoechst (Sigma). For MetO and Cyto c onthe one hand, and pTau on the other hand, 20 and 10 pictures with 20×and 40× magnifications respectively were taken per well using the InCellAnalyzer™ 1000 (GE Healthcare, France)

Analysis was done using Developer software (GE Healthcare) assessing theoverlap between MAP2 on one side, and MetO, Cyto c, Caspase 3 or pTaustaining on the other side. Results were expressed as the number ofoverlapping stained cells per field and reported as a percentage ofvehicle treated control.

Glutamate Release Assays:

After 4 h of Aβ₁₋₄₂ intoxication, cell media supernatants were analysedwith the Amplex Red Glutamic Acid assay kit (Invitrogen) according tothe manufacturer's instructions.

Results

Results presented in FIG. 21 show that the baclofen-acamprosatecombination corrects the major hallmarks of AD. Indeed, treatment withthe baclofen-acamprosate combination leads to a protection againstoxidative stress, as demonstrated by a significantly lower content inmethionine sulfoxide residues (MetO) in Aβ-intoxicated cells treatedwith baclofen-acamprosate when compared to non-treated intoxicated cells(FIG. 21 A). A marked reduction of the release of mitochondrialcytochrome C is also noticed in baclofen-acamprosate treatedAβ-intoxicated cells thereby showing that the combination is efficientin protecting the neuronal cells from Aβ induced apoptosis. (FIG. 21 B).Such effect was confirmed by the observation of a significant loweringof Caspase 3 upon baclofen-acamprosate treatment (not shown). Treatmentwith baclofen-acamprosate also significantly prevents Tauhyperphosphorylation in Aβ₁₋₄₂-treated neurons (FIG. 21 C), as well asglutamate accumulation in culture medium (FIG. 21 D), a featuremirroring excitotoxicity due to excess of glutamate in AD.

Baclofen-acamprosate combination is thus efficient in counteractingoxidative stress, apoptosis, hyperphosphorylated Tau accumulation, andglutamate excitotoxicity induced in neuronal cells by AB oligomers.These surprising properties can, at least in part, account for theobserved neuroprotective properties of the composition and for theactual correction of the disease. Such properties are of a particularinterest for the treatment of AD and related disorders, but also in thetreatment of other neurological disorders which share some of thesefeatures with AD. For instance, the control of the events leading to theaccumulation of pTau by the administration of the baclofen-acamprosatecombination is particularly relevant when considering the treatment ofTau pathologies such as frontotemporal dementia.

c) Protection Against the Toxicity of Aβ₁₋₄₂ in a Neurite Growth andSynapse Functionality Model.

Culture of Primary Cortical Neurons, Aβ₁₋₄₂ Oligomer Formation and CellIntoxication

Rat cortical neurons were cultured as described by Singer el al. [58].Briefly pregnant female rats of 15 days gestation were killed bycervical dislocation (Rats: Wistar) and the foetuses were removed fromthe uterus. The cortex was removed and placed in ice-cold medium ofLeibovitz (L15) containing 2% of Penicillin 10,000 U/ml and Streptomycin10 mg/ml and 1% of bovine serum albumin (BSA). Cortices were dissociatedby trypsin for 20 min at 37° C. (0.05%). The reaction was stopped by theaddition of Dulbecco's modified Eagle's medium (DMEM) containing DNaseIgrade II and 10% of foetal calf serum (FCS). Cells were thenmechanically dissociated by 3 serial passages through a 10 ml pipetteand centrifuged at 515×g for 10 min at +4° C. The supernatant wasdiscarded and the pellet of cells was re-suspended in a defined culturemedium consisting of Neurobasal supplemented with B27 (2%), L-glutamine(0.2 mM), 2% of PS solution and 10 ng/ml of BDNF. Viable cells werecounted in a Neubauer cytometer using the trypan blue exclusion test.The cells were seeded at a density of 30,000 cells/well in 96-wellplates (wells were pre-coated with poly-L-lysine (10 μg/ml)) and werecultured at +37° C. in a humidified air (95%)/CO₂ (5%) atmosphere.

After 10 days of culture, cells are incubated with drugs. After 1 hour,cells are intoxicated by 2.5 μM of beta-amyloid (1-42; Bachem) indefined medium without BDNF but together with drugs. Cortical neuronsare intoxicated for 24 hours. BDNF (10 ng/ml) is used as a positive(neuroprotective) control. Three independent cultures were performed percondition, 6 wells per condition.

Neurite Length and Synapse Quantitation

After 24 h of intoxication, the supernatant is taken off and thecortical neurons are fixed by a cold solution of ethanol (95%) andacetic acid (5%) for 5 min. After permeabilization with 0.1% of saponin,cells are blocked for 2 h with PBS containing 1% foetal calf serum.Then, cells are incubated with monoclonal antibody antimicrotubule-associated-protein 2 (MAP-2; Sigma) or withanti-synaptophysin (SYN, S5798, Sigma) together with anti-PSD95 (P246,Sigma) antibodies in order to quantify synapses. These antibodiesspecifically stain cell bodies and neurites of neurons (MAP2) or pre-and postsynaptic elements (SYN and PSD95, respectively).

These antibodies are revealed with Alexa Fluor 488 goat anti-mouse IgG(Molecular Probes). Nuclei of neurons were labeled by a fluorescentmarker (Hoechst solution, SIGMA).

Per well, 10 pictures are taken using InCell Analyzer™ 1000 (GEHealthcare) with 20× magnification. All pictures are taken in the sameconditions. Analysis of the neurite network is done using Developersoftware (GE Healthcare) in order to assess the total length of theneurite network.

Results

The combination of baclofen and acamprosate induces a significantprotective effect against the toxicity of human Aβ₁₋₄₂ peptide(improvement of 80% of neurite network) in primary cortical neuron cellsas shown in FIG. 7. The results clearly show that the intoxication byhuman amyloid peptide (Aβ₁₋₄₂ 2.5 μM) is significantly prevented by thecombination, whereas at those concentrations, baclofen or acamprosatealone have no significant effect on intoxication.

Furthermore, the total length of the neurite network in primary corticalneuron cells treated with this combination is no more significantlydifferent from control cells. Hence, this combination allows aneffective protection of cortical neuron cells against the toxicity ofhuman Aβ₁₋₄₂ peptide but also a neurite growth comparable to a sanecortical neuron cell.

d) Protection of Synapses of Hippocampal Neurons Against the Toxicity ofAβ₁₋₄₂ Oligomer Formation and Cell Intoxication.

Culture of primary hippocampal neurons, Aβ₁₋₄₂ oligomer formation andcell intoxication Hippocampus brain area is a key player in the buildingprocesses of memory; also, hippocampus atrophy is one of the mostaccurate pieces of evidence used in the diagnosis of AD. Aβ is known tohave a synaptotoxic activity which underlies the early cognitive declinein AD. The ability of combinations of the invention to protecthippocampal neuronal plasticity through protection of synapses fromAβ₁₋₄₂ toxicity has then been evaluated. Rat hippocampal neurons werecultured as described by Harrison (1990) [59]. Pregnant females (Wistar,Janvier) at 17 days of gestation were killed by cervical dislocation.Fetuses were collected and immediately placed in ice-cold Leibovitz(L15; Panbiotech) containing 2% of Penicillin 10,000 U/ml andStreptomycin 10 mg/ml (PS; Panbiotech) and 1% of bovine serum albumin(BSA; Panbiotech). Hippocampi were dissociated by trypsin (0.05%,Panbiotech) for 20 min at 37° C. The reaction had been stopped by theaddition of Dulbecco's modified Eagle's medium (DMEM; Panbiotech)containing DNAase I grade II (0.5 mg/ml; Panbiotech) and 10% of foetalcalf serum (FCS; Invitrogen). Cells were then mechanically dissociatedby 3 passages through a 10 ml pipette. Cells were centrifuged at 515 gfor 10 min at +4° C. The supernatant was discarded and pellet of cellswas re-suspended in a defined culture medium consisting of Neurobasal(Invitrogen) supplemented with B27 (2%; Invitrogen), L-glutamine (2 mM;Panbiotech), 2% of PS solution and 10 ng/ml of brain-derivedneurotrophic factor (BDNF, Panbiotech). Viable cells were counted in aNeubauer cytometer, using the trypan blue exclusion test, then seeded ata density of 20,000 per well in a 96-well plates pre-coated withpoly-L-Lysine (Greiner) and cultured at 37° C. in an air (95%)-CO2 (5%)incubator. The medium was changed every 2 days.

After 18 days of culture, cells were incubated with R/S baclofen andacamprosate combination (80 nM and 0.32 nM respectively). After 2 daysof culture, hippocampal neurons were intoxicated with Aβ₁₋₄₂ humanpeptide (Bachem) at 0.3 μM during 48 hours in the presence of the drugcombination.

BDNF (50 ng/ml) has been used as positive control and referencecompound.

Three separate cultures were performed per condition, 6 wells percondition.

PSD95 and Synaptophysin Immunostaining, Synaptic Loss Quantification.

After 48 hours of intoxication, cells were permeabilized andnon-specific sites were blocked with a solution of PBS containing 0.1%of saponin and 1% of FCS for 15 min and then were incubated with mousemonoclonal primary antibody against Post Synaptic Density 95 kDa (PSD95,Abcam) and with rabbit polyclonal primary antibody against Synaptophysin(Sigma) overnight at 4° C. These antibodies were revealed with AlexaFluor 488 goat anti-mouse (Molecular probe) and Alexa Fluor 568 goatanti-rabbit (Molecular probe) for 1 hour. Nuclei of cells were labelledby a fluorescent marker (Hoechst solution, SIGMA). The total area ofPSD95, Synaptophysin and the colocalization were evaluated. Results wereexpressed in μm² per field. For each well of culture, 40 pictures perwell were taken using InCell Analyzer™ 2000 (GE Healthcare) with 60×magnification. Colocalization of the two labellings corresponds tointact synapses. The area was automatically evaluated with Developersystem analysis (GE Healthcare). PSD95 and synaptophysin total surfaceoverlap was quantified (in μm²) and computed for each condition.

Data were expressed in percentage of control conditions (nointoxication, no Aβ₁₋₄₂=100%) in order to express the amyloid injury.All values were expressed as mean+/−SEM (s.e. mean). Statisticalanalyses were done on the different conditions (ANOVA followed byDunnett's test or t-test).

Results

Results show that Aβ₁₋₄₂ (0.3 μM) intoxication induces a significantlowering of PSD95 and synaptophysin colocalization surface (almost 34%,FIG. 20, black bar) when compared to non-intoxicated cells cultures.Incubation with the baclofen-acamprosate (80 nM and 0.32 nMrespectively) combination is found to significantly reverse Aβ₁₋₄₂oligomer toxicity toward synapse junctions: a 62% increase incolocalization area is observed when compared to non-treated cells (FIG.20, light grey bar). Hence the baclofen-acamprosate combination isefficient in maintaining synaptic junctions between hippocampal cellseven in the presence of Aβ oligomers.

Baclofen-acamprosate therapy is thus efficient in maintaining synapsefunction in the presence of Aβ oligomers; together with the neuritisgrowth protection as mentioned above, this therapy can be thusconsidered as of particular interest for protecting synaptic plasticityand cellular networking which is impaired in AD.

e) Molecular Targets Important for Baclofen-Acamprosate Mechanism ofAction in Neuroprotection Against Aβ Toxicity.

Culture of Primary Cortical Neurons, Ligand Cell Intoxication

Culture of rat primary cortical cells, Aβ₁₋₄₂ peptide preparation andcell intoxication were performed as stated in I.A.2.a). Non-toxicconcentrations of CGP54626 (10 μM, GABABR antagonist, TocrisBiosciences), muscimol (1 μM, GABA_(A)R agonist, Sigma Aldrich),strychnine (2.5 μM, Glycine receptor antagonist, Tocris Biosciences),(S)-3,5-dihydroxyphenylglycine (DHPG, mGluR1/5 agonist 10 μM, TocrisBiosciences) and (2R,4R)-amino-2,4-pyrrolidinedicarboxylic acid (APDC,mGluR2/3 agonist, 0.3 μM, Sigma Aldrich) were dissolved in 0.1% DMSO(except for DHPG dissolved in water) and added 2 h (except for DHPG, 1h) before ACP (8 nM) or BCL (400 nM) to 11 days rat primary neuronalcell cultures.

BDNF at 50 ng/ml was used as a positive control.

Cell Survival Evaluation After 24 hours incubation with Aβ₁₋₄₂ peptide,cortical neurons were fixed by a cold solution of alcohol/acetic acidduring 5 minutes. Then, cells were permeabilized and non-specific siteswere blocked with a solution of PBS containing 0.1% of saponin (Sigma)and 1% of fetal calf serum for 15 min. Cells were then incubated withmouse monoclonal primary microtubule-associated protein 2 antibody(MAP-2, Sigma) for 2 hours in the same solution at the dilution of1/400. An incubation of 1 hour with Alexa Fluor 488 goat anti-mouse(Molecular Probes) at 1/400 as a secondary antibody was thereafterperformed. Nuclei of cells were labelled by a fluorescent marker(Hoechst solution, Sigma).

Total neuronal survival was evaluated by numbering MAP-2 positiveneuronal cell bodies. Ten pictures per well of culture were taken usingInCell Analyzer™ 2000 (GE Healthcare) with 20× magnification. The numberof neurons was automatically evaluated with Developer system analysis(GE Healthcare).

Data are expressed in percentage of control conditions (no intoxication,no amyloid=100%) in order to express the amyloid injury. All values areexpressed as mean+/−SEM (s.e. mean). An ANOVA followed by Dunnett's testwas done on each condition.

Results

Data gathered in these experiments show that CGP54626, an orthostericantagonist of GABA_(B) receptors, blocks, at non-toxic doses, theneuroprotective effect of baclofen (not shown), which confirms theimportance of GABA_(B) receptor activation for the neuroprotectiveaction of baclofen within baclofen-acamprosate combination. In addition,muscimol, an agonist of GABA_(A) receptors, was found to block theneuroprotective effect of acamprosate (FIG. 22 A), which demonstratesthe role of the antagonistic activity of acamprosate on GABA_(A)receptors for the protection of neuronal cultures against Aβ₁₋₄₂cytotoxicity. An agonistic effect on ionotropic glycine receptors isalso found necessary for neuroprotection since strychnine, an antagonistof inhibitory glycine-gated channels, reverses the neuroprotectiveeffect of the acamprosate (FIG. 22 B). Preincubation with DHPG and APDC,agonists of group I and II metabotropic glutamate receptorsrespectively, results in the abolition of neuroprotection normallyafforded by acamprosate, which demonstrates the importance of theantagonistic effect of acamprosate on these molecular targets for itsneuroprotective effect (FIGS. 22 C and D).

From the above, it can be deduced that the particularly effectiveneuroprotective effect reported for the baclofen-acamprosate combinationis the result of a concerted action on, at least, the above 5 moleculartargets. Acamprosate is moreover of particular interest because of itssimultaneous actions on at least GABA_(A) receptors and ionotropicglycine receptors as well as group I and II metabotropic glutamatereceptors which, as demonstrated above, play an essential role inneuroprotection against Aβ peptides.

3. Concentration Ranges of Baclofen-Acamprosate Combination afford aProtection in the Three Main Features of Aβ Peptide Toxicity.

As mentioned in the set of the above experiments, the inventors havefound that the baclofen-acamprosate combination is efficient incounteracting events triggered by the oligomers of Aβ at the synaptic,at the neuronal and at the endothelial levels.

The inventors have been able to determine ranges of concentrations ofbaclofen and acamprosate that allow an effective protection, at the sametime, of neurons, synapse function and endothelial function (FIG. 23).Such a range for a simultaneous activity has been determined for thesethree features to be from 80 nM to 1 μM for baclofen and from 320 μM to4 nM for acamprosate.

A particularly enhanced efficacy of the treatment can then be expectedwithin these ranges of concentrations due to the conjunction of effectsthat is obtained. It can be thus considered that reaching these rangesof plasmatic concentrations of baclofen and acamprosate in the brain isof particular interest.

4. Examples of Synergistic Combinations

The inventors have further found that the baclofen-acamprosatecombination is active and shows a marked synergistic effect according tomethods commonly recognized among pharmacology community [60-62] in atleast one of the above in vitro models for AD (Table 6) in the testedconcentrations. This ensures a particular clinical benefit. At certainconcentrations, the combination provides a synergistic protection in allthree models, which is even more particularly advantageous.

TABLE 6 Combination activity ACP/BCL Neuritis (concentration, M)Angiogenesis Neuroprotection network 1.44 10⁻¹⁰/2.00 10⁻⁰⁶ Synergy ¥Synergy 1.44 10⁻¹⁰/3.60 10⁻⁰⁸ Synergy Synergy Synergy 1.44 10⁻¹⁰/4.0010⁻⁰⁷ ‡ Synergy Synergy 1.44 10⁻¹⁰/8.00 10⁻⁰⁸ Synergy Synergy Synergy3.20 10⁻¹⁰/2.00 10⁻⁰⁶ Synergy ¥ Synergy 3.20 10⁻¹⁰/3.60 10⁻⁰⁸ SynergySynergy Synergy 3.20 10⁻¹⁰/4.00 10⁻⁰⁷ Synergy Synergy Synergy 3.2010⁻¹⁰/8.00 10⁻⁰⁸ Synergy Synergy Synergy 1.60 10⁻⁰⁹/1.60 10⁻⁰⁸ ‡ SynergyNA 1.60 10⁻⁰⁹/3.60 10⁻⁰⁸ ‡ Synergy Synergy 1.60 10⁻⁰⁹/4.00 10⁻⁰⁷ SynergySynergy Synergy 1.60 10⁻⁰⁹/8.00 10⁻⁰⁸ Synergy Synergy Synergy 6.4010⁻¹¹/1.60 10⁻⁰⁸ Synergy Synergy NA 6.40 10⁻¹¹/2.00 10⁻⁰⁶ Synergy ¥ NA6.40 10⁻¹¹/3.60 10⁻⁰⁸ Synergy Synergy NA 6.40 10⁻¹¹/4.00 10⁻⁰⁷ ‡ SynergyNA 6.40 10⁻¹¹/8.00 10⁻⁰⁸ Synergy Synergy NA 6.40 10⁻¹¹/3.20 10⁻⁰⁹ NA NASynergy 8.00 10⁻⁰⁹/3.60 10⁻⁰⁸ ‡ ¥ Synergy Synergy is determinedaccording Loewe or Bliss methods [60-62] ‡: no effect; ¥: no synergy;NA: not available

B. Baclofen-Acamprosate Combination Therapies Prevent Toxicity of HumanAβ in In Vivo Models

1. Intracerebroventricular Administration of Aβ₂₅₋₃₅ in Swiss Mice.

Animals

Male Swiss mice, 6 weeks old, are used throughout the study. Animals arehoused in plastic cages, with free access to laboratory chow and water,except during behavioural experiments, and kept in a regulatedenvironment, under a 12 h light/dark cycle (light on at 8:00 a.m.).Experiments are carried out in a soundproof and air-regulatedexperimental room, to which mice have been habituated at least 30 minbefore each experiment.

Combinatory treatment and administration of Aβ₂₅₋₃₅

Drug(s) is/are daily administered by gavage (per os). The Aβ₂₅₋₃₅peptide and scrambled Aβ₂₅₋₃₅ peptide (control) have been dissolved insterile bidistilled water, and stored at −20° C. until use. Theβ-amyloid peptides are then administered intracerebroventricularly(i.c.v.). In brief, each mouse is anaesthetized lightly with ether, anda gauge stainless-steel needle is inserted unilaterally 1 mm to theright of the midline point equidistant from each eye, at an equaldistance between the eyes and the ears and perpendicular to the plane ofthe skull. Peptides or vehicle are delivered gradually withinapproximately 3 s. Mice exhibit normal behaviour within 1 min afterinjection. The administration site is checked by injecting Indian ink inpreliminary experiments. Neither insertion of the needle nor injectionof the vehicle have a significant influence on survival, behavioralresponses or cognitive functions.

On Day −1, i.e., 24 h before the Aβ₂₅₋₃₅ peptide injection, baclofen,acamprosate, a combination thereof or the vehicle solution areadministered twice per os by gavage at 8:00 am and 6:00 pm.

On Day 0 (at 10:00 am), mice are injected i.c.v. with Aβ₂₅₋₃₅ peptide orscrambled Aβ₂₅₋₃₅ peptide (control) in a final volume of 3 μl (3 mM).

Between Day 0 and Day 7, baclofen, acamprosate, a combination thereof orthe vehicle solution are administered per os by gavage twice daily (at8:00 am and 6:00 pm). A dose designated as bid (bis in die) means thatsaid dose is administered twice daily. Drugs are solubilized in waterand freshly prepared just before each gavage administration. One animalgroup receives donepezil (reference compound, 1 mg/kg/day) in a singleinjection intraperitoneally (at 8:00 am).

On Day 7, all animals are tested for the spontaneous alternationperformance in the Y-maze test, an index of spatial working memory.

On Days 7 and 8, the contextual long-term memory of the animals isassessed using the step-down type passive avoidance procedure.

On Day 8, animals are sacrificed. Their brains are dissected and kept at−80° C. for further analysis.

When the baclofen-acamprosate combination is tested in a combinationwith reference compounds for AD (for instance, currently approvedtreatments for AD are donepezil, galantamine, rivastigmine ormemantine), said reference compounds are administered intraperitoneally,between Day 0 and Day 7, at 8:00 am.

a) Combinations Enhance Behavioral and Cognitive Performances ofIntoxicated Animals

Spontaneous Alternation Performances Y-Maze Test

On Day 7, all animals are tested for spontaneous alternation performancein the Y-maze, an index of spatial working memory. The Y-maze is made ofgrey polyvinylchloride. Each arm is 40 cm long, 13 cm high, 3 cm wide atthe bottom, 10 cm wide at the top, and converges at an equal angle. Eachmouse is placed at the end of one arm and allowed to move freely throughthe maze during an 8 min session. The series of arm entries, includingpossible returns into the same arm, are checked visually. An alternationis defined as entries into all three arms on consecutive occasions. Thenumber of maximum alternations is therefore the total number of armentries minus two and the percentage of alternation is calculated as(actual alternations/maximum alternations)×100. Parameters include thepercentage of alternation (memory index) and total number of arm entries(exploration index). Animals that show an extreme behavior (Alternationpercentage<25% or >85% or number of arm entries <10) are discarded.Usually, this accounts for 0-5% of the animals. This test incidentallyserves to analyze at the behavioral level the impact and the amnesiceffect induced in mice by the Aβ₂₅₋₃₅ injection.

Passive Avoidance Test

The apparatus is a two-compartment (15×20×15 cm high) box with oneilluminated with white polyvinylchloride walls and the other darkenedwith black polyvinylchloride walls and a grid floor. A guillotine doorseparates each compartment. A 60 W lamp positioned 40 cm above theapparatus lights up the white compartment during the experiment.Scrambled foot shocks (0.3 mA for 3 s) could be delivered to the gridfloor using a shock generator scrambler (Lafayette Instruments,Lafayette, USA). The guillotine door is initially closed during thetraining session. Each mouse is placed into the white compartment. After5 s, the door raises. When the mouse enters the darkened compartment andplaces all its paws on the grid floor, the door closes and the footshock is delivered for 3 s. The step-through latency, that is, thelatency spent to enter the darkened compartment, and the number ofvocalizations is recorded. The retention test is carried out 24 h aftertraining. Each mouse is placed again into the white compartment. After 5s the door is raised, the step-through latency and the escape latency,i.e., the time spent to return into the white compartment, are recordedup to 300 s.

Results

Positive results are observed in behavioural performances andbiochemical assays performed 7 days after Aβ₂₅₋₃₅ peptide i.c.v.injection.

The combination of baclofen and acamprosate induces a significantprotective effect on behavioral and cognitive performances ofintoxicated animals as shown in FIGS. 8, 9 and 10.

In FIG. 8, with only 53.8% of alternation, intoxicated mice exhibit astrongly impaired spatial working memory compared to control. With animprovement of more than 48% of their percentage of alternation comparedto intoxicated controls, the impairment is significantly prevented inmice treated with baclofen and acamprosate.

Similarly, FIGS. 9 and 10 show that intoxicated animals exhibit impairedbehavioral and cognitive performances according to their scores inescape latency and step-through latency respectively. In both tests, thecombination of baclofen and acamprosate allows a significant correctionof the impairment. The escape latency of mice treated with thiscombination is no more significantly different from control mice (FIG.9) and step-through latency (FIG. 10) is significantly increased bycombinations of the invention, with an enhanced effect of thecombination compared to the drugs alone.

Memory impairment is the early feature of Alzheimer's disease and theseresults clearly show that the toxic effect of amyloid peptide onbehavioral and cognitive performances (including memory) issignificantly prevented by the combinations of the invention.

Furthermore, FIG. 16 shows that extremely low doses of baclofen (480μg/kg bid), acamprosate (32 μg/kg bid) and donepezil (0.25 mg/kg/day)can be combined to allow complete protection of behavioral and cognitiveperformances of mice as measured by the Y-maze test. While donepezil, atthis concentration, has no significant effect (32% protection) onspatial working memory, its use in conjunction with the baclofen andacamprosate combination allows a complete protection (98%) ofintoxicated mice's cognitive performances. The combination index ofdonepezil in combination with baclofen-acamprosate is 0.687 whichdetermines a marked synergy between the compounds (as determined by themethod of Loewe [60, 61]). The human equivalent dose of donepezil usedin this combination is more than 4 times lower than the lowest andalmost 20 times lower than the highest dose currently used in humans forAD treatment.

An improvement is also observed in the performances in the Y-maze testwhen combining baclofen (480 μg/kg bid), acamprosate (32 μg/kg bid) andmemantine (0.5 mg/kg/day), as shown in FIG. 19. At the concentrationsused neither the baclofen-acamprosate combination nor memantine has asignificant effect. Noteworthy, the dose of memantine used in theseexperiments is more than 7 times lower than the human equivalent dosecorresponding to the maintenance treatment. Moreover the combination ofbaclofen-acamprosate with memantine is synergistic, displaying acombinatory index of 0.784 (as determined by the method of Loewe [60,61]).

Combinations of the invention can thus be further combined with othertherapies for AD in order to potentiate their action, and to lower theirpotential side effects by using lower doses for these drugs.

b) Combinations Improve Neurophysiological Concern of NeurologicalDiseases

Combination therapies are tested in an in vivo model of Aβ intoxication.Their effects on several parameters which are affected in neurologicaldiseases are assessed:

-   -   Caspases 3 and 9 expression level, considered as an indicator of        apoptosis,    -   Lipid peroxidation, considered as a marker for oxidative stress        level,    -   GFAP expression assay, considered as a marker of the level of        brain inflammation,    -   Blood-Brain Barrier integrity,    -   Overall synapse integrity (synaptophysin ELISA), and    -   Quantification of viable neurons in the Cornus Ammonis area1        (CA1) of hippocampus.

Blood-Brain Barrier Integrity

The experimental design about animal intoxication by Aβ is the same aspreviously stated.

The potential protective effect of the combination therapies onblood-brain barrier (BBB) integrity is analyzed in mice injectedintracerebroventricularly (i.c.v.) with oligomeric amyloid-β25-35peptide (Aβ₂₅₋₃₅) or scrambled Aβ₂₅₋₃₅ control peptide (Sc.Aβ), 7 daysafter injection.

On day 7 after the Aβ₂₅₋₃₅ injection, animals are tested to determineBBB integrity by using the EB (Evans Blue) method. EB dye is known tobind to serum albumin after peripheral injection and has been used as atracer for serum albumin. EB dye (2% in saline, 4 ml/kg) is injectedintraperitoneally (i.p.) 3 h prior to the transcardiac perfusion. Miceare then anesthetized with i.p. 200 μl of pre-mix ketamine 80 mg/kg,xylazine 10 mg/kg, and the chests are opened. Mice are perfusedtranscardially with 250 ml of saline for approximately 15 min until thefluid from the right atrium becomes colourless. After decapitation, thebrains are removed and dissected out into three regions: cerebral cortex(left+right), hippocampus (left+right), diencephalon. Then, each brainregion is weighed for quantitative measurement of EB-albuminextravasation.

Samples are homogenized in phosphate-buffered saline solution and mixedby vortexing after addition of 60% trichloroacetic acid to precipitatethe protein. Samples are cooled at 4° C., and then centrifuged 30 min at10,000 g, 4° C. The supernatant is measured at 610 nm for absorbance ofEB using a spectrophotometer.

EB is quantified both as:

-   -   μg/mg of brain tissue by using a standard curve, obtained by        known concentration of EB-albumin, and    -   μg/mg of protein.

Overall Synapse Integrity (Synaptophysin ELISA)

Synaptophysin has been chosen as a marker of synapse integrity and isassayed using a commercial ELISA kit (USCN, Ref. E90425Mu). Samples areprepared from hippocampus tissues and homogenized in an extractionbuffer specific to as described by manufacturer and referenceliterature.

Tissues are rinsed in ice-cold PBS (0.02 mol/1, pH 7.0-7.2) to removeexcess blood thoroughly and weighed before nitrogen freezing and −80° C.storage. Tissues are cut into small pieces and homogenized in 1 mlice-cold phosphate buffer saline (PBS) solution with a glasshomogenizer. The resulting suspension is sonicated with an ultrasoniccell disrupter or subjected to two freeze-thawing cycles to furtherbreak the cell membranes. Then, homogenates are centrifugated for 5 minat 5,000 g and the supernatant is assayed immediately.

All samples are assayed in triplicate.

Quantification of proteins is performed with the Pierce BCA(bicinchoninic acid) protein assay kit (Pierce, Ref. 23227) to evaluateextraction performance and allow normalization.

The total protein concentrations are then calculated from standard curvedilutions and serve to normalize ELISA results.

Quantification of Viable Neurons in the CA1

On Day 8, each mouse is anesthetized with 200 μl i.p. of a pre-mix ofketamine 80 mg/kg and xylazine 10 mg/kg and transcardially perfused with100 ml of saline solution followed by 100 ml of 4% paraformaldehyde. Thebrains are removed and kept for 24 h post-fixation in 4%paraformaldehyde solution at 4° C. After post-fixation, brains arewashed in a phosphate buffer saline (PBS) solution, then cerebellums areremoved and the brains are cut in coronal sections (20 μm thickness)using a vibratome (Leica VT100OS, Leica, Wetzlar, Germany). Serialsections are placed on 24-well plates with PBS. They are then selectedto include the hippocampal formation and 9 sections are placed ingelatin-coated glass strips (one slide per animal for cresyl violet).All slides are dried at room temperature for 48 h to avoid unsticking.The slides are stored at room temperature until cresyl violet staining.Sections are stained with 0.2% cresyl violet reagent (Sigma-Aldrich),then dehydrated with graded ethanol, treated with toluene, and mountedwith Mountex medium (BDH Laboratory Supplies, Poole, Dorset, UK).

After mounting, slides are kept at RT for 24 h drying. Examination ofthe CA1 area is performed using a light microscope (Dialux 22, Leitz),with slices digitalized through a CCD camera (Sony XC-77CE, Sony, Paris,France) with the NIH Image® v1.63 software (NIH). CA1 measurement andpyramidal cells counts are processed using ImageJ® (NIH). Data areexpressed as mean of nine slices of CA1 pyramidal cells per millimeterfor each group (left and right hippocampus CA1 counting) [63].

Oxidative Stress Assay

Mice are sacrificed by decapitation and both hippocampi are rapidlyremoved, weighed and kept in liquid nitrogen until assayed. Afterthawing, hippocampi are homogenized in cold methanol (1/10 w/v),centrifuged at 1,000 g during 5 min and the supernatant placed inEppendorf tubes. The reaction volume of each homogenate is added toFeSO4 1 mM, H2SO4 0.25 M, xylenol orange 1 mM and incubated for 30 minat room temperature. After reading the absorbance at 580 nm (A580 1), 10A1 of cumene hydroperoxyde 1 mM (CHP) is added to the sample andincubated for 30 min at room temperature, to determine the maximaloxidation level. The absorbance is measured at 580 nm (A580 2). Thelevel of lipid peroxidation is determined as CHP equivalents (CHPE)according to: CHPE=A580 1/A580 2×[CHP] and expressed as CHP equivalentsper weight of tissue and as percentage of control group data.

Caspase Pathway Induction Assay and GFAP Expression Assay

Mice are sacrificed by decapitation and both hippocampi are rapidlyremoved, rinsed in ice-cold PBS (0.02 mol/l, pH 7.0-7.2) to removeexcess blood thoroughly, weighed and kept in liquid nitrogen untilassayed. Tissues are cut into small pieces and homogenized in 1 mlice-cold PBS with a glass homogenizer. The resulting suspension issonicated with an ultrasonic cell disrupter or subjected to twofreeze-thawing cycles to further break the cell membranes. Then,homogenates are centrifugated at 5,000 g during 5 min and thesupernatant is assayed immediately.

Experiments are conducted with commercial assays: Caspase-3(USCN—E90626Mu), Caspase-9 (USCN—E90627Mu). GFAP (USCN—E90068).

Quantification of proteins is performed with the Pierce BCA(bicinchoninic acid) protein assay kit (Pierce, Ref. 23227) to evaluateextraction performance and allow normalization.

Results

The combination of baclofen and acamprosate induces a significantprotective effect on neurophysiological functions of intoxicated animalsas shown in FIGS. 11, 12, 13 and 14.

With a protection of more than 60% compared to non-treated intoxicatedanimals, the combination is effective for the protection of neurons(FIG. 11) and synaptic density (FIG. 13).

Similarly, FIG. 12 shows that the combination of baclofen andacamprosate protects the BBB integrity (76%) compared with non-treatedintoxicated animals.

Finally, this combination therapy is efficient in reducing the overalloxidative stress induced by Aβ in the brains of treated animals whencompared with non-treated intoxicated animals (FIG. 14).

2. hAPPSL Transgenic Mice

Efficiency of the baclofen-acamprosate combination has been tested inanother murine model for AD. A mouse line overexpressing the 751 aminoacid form of human APP (hAPP) with London (V717I) and Swedish(KM670/671NL) mutations (hAPP_(SL)) under the control of the murineThy-1 promoter has been chosen. In this model, significant deficits inspatial memory and learning in the water maze task and in habituation inthe hole-board task are noticed as soon as 6 months of age. Thiscognitive decline is due to dysfunction of synaptic transmission andmimics some aspects of the early phases of human AD.

Animals

Experiments were conducted on male subjects. Animals were bred at QPS(Austria), and C57BL6 age and gender-matched transgenic littermatesserved as controls. Animals were housed in plastic cages with freeaccess to food and water, except during behavioural experiments, andkept in a regulated environment (23±1° C., 50-60% humidity) under a 12 hlight/dark cycle.

Drug Treatment

Drug combination (acamprosate 0.2 mg/kg and baclofen 3 mg/kg) or thevehicle solution are administered per os by gavage twice daily (at 8:00am and 6:00 pm) in a volume of 5 ml/kg. Treatment with thebaclofen-acamprosate combination was started at 8 months of age (i.e.,when the disease is well established) and lasted 4 weeks until the endof functional tests. All behavioral tests were performed 2 h aftertreatment.

Morris Water Maze (MWM) Acquisition Task

Mice were tested for acquisition and working memory. Briefly, swimmingwas recorded using a Videotrack software (Viewpoint,Champagne-au-Mont-d'Or, France), with trajectories being analysed aslatencies and distances. The software divides the pool into fourquadrants. Training (Acquisition) consisted of 4 swims per day for 4days. The latency, expressed as mean±s.e.m., was calculated for eachtraining day. A probe test was performed 24 h after the last swim(retention phase). The platform was removed and each animal was alloweda free 60 s swim. The time spent in each quadrant was then determined.After the training test, animals were tested for spatial working memory.Working memory was specifically assayed by changing the platformlocation every day (four swims per day during 2 days) and by using atraining inter-trial time of 2 min. The swimming times to find theplatform of the first (swim1), second (swim2), third (swim3) and fourthtrial (swim4) of Day 2 were calculated and averaged.

Statistical Analyses

A mixed ANOVA with Dunnett's test, including fixed effect terms fortreatment, time and the treatment by time interaction, and a randomeffect term for animals was applied. Treatment effect was assessed foreach time point and for combined time points (global effect). Tests wereconducted at a 5% significance level.

Results

At the age of 8 months, when hAPP_(SL) transgenic mice's cognition wasalready impaired and diseased installed, a one-month treatment with thebaclofen-acamprosate combination is found to substantially improvecognitive deficits as they had been assessed in the MWM acquisition andworking memory tests (FIGS. 24 A) and B) respectively).

Thus, notwithstanding the extensive in vitro results, the resultsobtained in two different in vivo models confirm the efficacy of thecombination in counteracting the toxicity of Aβ and its behaviouraleffects as well as its effects on brain physiology.

Several neurological functions impaired in numerous neurologicaldisorders, including neurodegenerative disorders such as Alzheimer'sdisease and related disorders, have been protected and symptoms retardedor reduced by the combination of baclofen-acamprosate.

C. Baclofen-Acamprosate Combination Therapies have Positive Effects inHuman Subjects

The particular efficiency of the compositions of the invention indifferent in vitro and animal models for AD, together with thepossibility of acting not only on a single but on a group of phenomenadescribed as being at the origin of the disease, have prompted theinventors to test it in aged people, in a model of chemically inducedamnesia that is commonly used in clinical trials for AD, and also inpeople suffering from mild AD. More particularly, baclofen-acamprosatecombination efficacy in improving memory or memory related mentalfunctions and in improving electrophysiological features underlyingworking memory has been assessed through clinical trials.

The studies were conducted in compliance with the following protocolsand in accordance with Good Clinical Practice (GCP) as required by theEuropean Medicines Agency ICH-E6 (RI) guideline recommendations and theFrench law No. 2004-806, Aug. 9, 2004 relative to public health law.

1. Improvement in Memory and Related Functions in Humans Subjected toChemically-Induced Cognitive Impairment.

AD patients show the signs of diminished cholinergic synaptic activity,with diminished levels of acetylcholine. The pharmacological model ofthis phenomenon consists of treating the subjects with scopolamine, ablocker of muscarinic receptors. This drug thereby induces a transitoryand reversible cognitive impairment upon administration [64]; it istherefore currently used as a pharmacologic model for induced dementia.

Experimental Design

Twenty-one healthy male volunteers aged from 20 to 40 years wereenrolled in the study, which is a randomized, 2-way cross-over, doubleblind, placebo-controlled study. The study had two distinct periods, P1and P2, each consisting of 40 hours hospitalization and being spacedfrom each other by 7 days as a wash-out period. During both theseperiods, the baclofen-acamprosate combination is administered orally,the drugs being administered concomitantly. Results were compared tothose of placebo treatment. A dose designated as bid (bis in die) meansthat said dose is administered twice daily.

During P1, subjects were administered according to the followingtreatment.

-   -   at Day 1: baclofen (6 mg) and acamprosate (0.4 mg) mix, or        placebo, orally, in a sub-acute administration, bid.    -   at Day 2 (test day), H3 just before scopolamine injection:        single dose of baclofen (6 mg) and acamprosate (0.4 mg) mix or        placebo, orally.    -   at Day 2, H3: a sub-cutaneous injection of 0.5 mg of        scopolamine.

According to the same above dosage schedule, subjects who had receivedbaclofen-acamprosate treatment in P1 received placebo during P2, whilethose administered with placebo in P1 were treated with thebaclofen-acamprosate mix in P2.

Cognitive Test

Effects on impaired cognition were explored by measuring the followingfeatures in the Cognitive Groton Maze Learning Task (GMLT) test:

-   -   the efficiency of performance: mps,    -   the total number of errors: ter, and    -   the duration of task: dur.

The GMLT test was performed for each treatment period on Day 1 (2training sessions), on Day 2 (test day) HO, H2.5, H4, H5.5, H7 and H9and on Day 3 H24.

Data Analyses

The data from the GMLT test were pooled together in a “composite” GMLTscore. Comparison between treatment groups for the two periods wasperformed on change from baseline value by using a mixed Analysis ofCovariance (ANCOVA) model with SAS® Mixed procedure (see WorldwideWebsite sas.com), including fixed effect terms for period treatment,time and the treatment by time interaction, a random effect term forsubject and the baseline value as covariate. Treatment effect wasassessed for combined time points (global effect) and for each timepoint.

Results: Baclofen-Acamprosate Combination Improves the Cognitive DeficitInduced by Scopolamine.

For each of the periods P1 and P2, similar data were obtained for eachof mps, ter and dur components of the GMLT test. The resulting“composite” GMLT score is presented in FIG. 25.

Scopolamine is known to act as early as 30 min after its administration,its effect lasting for approximately 6 hours. Its Tmax is known to be ofabout 3 hours [64]. These data correspond to the transient collapse ofperformances in the GMLT test observed in placebo-treated subjects (FIG.25, circles, dotted line).

An improvement of cognitive performances was observed forbaclofen-acamprosate treated subjects (FIG. 25 squares, grey line)compared to placebo dosed subjects. This improvement is particularlysignificant in the time period around H5.5 that corresponds to the Tmaxof baclofen-acamprosate (FIG. 25, darkest area in the pharmacokineticscale of mix compounds).

Hence, the baclofen-acamprosate combination is efficient incounteracting scopolamine induced cognitive impairment. Hence, besidesbeing considered as of interest in the cases of provoked amnesia,combinations of the invention can also be considered as of particularinterest in reversing memory impairment related to conditions implyingdiminished cholinergic synaptic activity. Baclofen-acamprosatecombinations are thus of particular interest for the treatment of AD andrelated diseases.

2. Baclofen-Acamprosate is Effective in Mild AD Patients.

Another part of the studies performed on human subjects consists of athree-month clinical study that is currently performed on patientsdiagnosed with mild AD with the aim of exploring the effect ofbaclofen-acamprosate combinations on cognitive and behaviouralimpairments related to the disease. This study is a 12-week, prospectivepilot single-blind and placebo sequential controlled, multi-center trialwhich assesses the effects of different doses of thebaclofen-acamprosate combination. It is a“challenge/de-challenge/re-challenge” (CDR) type study, which means thatthe baclofen-acamprosate combination is administered on a given timeperiod, then withdrawn, and then re-administered. During the withdrawal(de-challenge) phase, the medication is allowed to wash out of theorganism. Then, an improvement of the disease's symptoms during thechallenge and re-challenge phases, together with no improvement or aworsening in the de-challenge phase, would be considered as a signatureof the efficiency of the combination. This study is conducted in an openway and was single-blind for the patients and the neurophysiologistassessor. Preliminary results obtained for 29 patients are presentedherein.

Experimental Design

Cognitive and behavioural impairments of the subjects are measured bypsychometric assessments during visits to a neurophysiologist set at thebeginning and the end of each of the 3 phases of the CDR study:

Visit 1 (V1) holds on the beginning of the challenge phase, which lasts4 weeks, and at the end of which performances of subjects are evaluatedduring visit 2 (V2); then the de-challenge phase of 4 weeks runs andends with visit 3 (V3), after which the re-challenge phase lasts 4 weeksmore, to be ended by the last evaluation of the subjects by theneurophysiologist during visit 4 (V4). At each of visits V1-V4, ADAS cogtests (Alzheimer's Disease Assessment Scale-cognitive subscale) areperformed to evaluate the changes in cognition performances of thesubjects.

In this study, three doses, administered twice daily (once in themorning and once in the evening), of the mix of baclofen-acamprosate aretested:

-   -   Dose 1: 0.4 mg acamprosate and 6 mg baclofen, given        concomitantly;    -   Dose 2: 1 mg acamprosate and 15 mg baclofen, given        concomitantly; and    -   Dose 3: 20 mg acamprosate and 12 mg baclofen, given        concomitantly.

Subject Recruitment

29 patients diagnosed as suffering from mild AD, i.e., displaying a MiniMental State Examination (MMSE) score between 20 and 26, were enrolledin the study (clinical characteristics of the cohort, Table 7) for thetesting of doses 1 and 2.

TABLE 7 n = 29 patients Age +/− sem 71.8 +/− 1.33 years Gender 13Females 16 Males MMSE +/− sem 23.4 +/− 0.389 ADAS Cog. at V1 +/− sem11.9 +/− 0.661 sem: standard error of the mean; MMSE: Mini Mental StateExamination; ADAS cog: Alzheimer's Disease Assessment Scale-cognitivesubscale

Depending on the subjects, the screening/recruitment sessions were heldfrom 1 to 2 weeks before V1.

At V1, the 29 patients were randomized into two groups:

-   -   15 patients in group 1 receiving dose 1, and    -   14 patients in group 2 receiving dose 2.

ADAS cog score assessed at V1 is taken as the baseline to evaluatechanges in performances of the subjects assessed during subsequentvisits V2, V3 and V4.

Each patient is their own control for the 3 successive sequences of theCDR study.

The effect of the mix is assessed on the changes of ADAS cog score atthe end of each period in comparison with the previous visit and thetreatment periods are compared to the placebo period.

The mean changes from baseline to the end of the first 4-week activetreatment period and the mean changes observed between each visit areassessed.

The change of each endpoint is measured between each visit:

-   -   from baseline (V1) to the end of the first 4-week active        treatment period (V2),    -   from V2 to the end of the 4-week placebo period (V3),    -   from V3 to the end of the second 4-week active treatment period        (V4), and    -   from V1 to V4.

Differences between V1 and V2 or V1 and V4 are statistically testedusing a one-tailed paired test of Student at a 10% significance level.

Dosing of Plasmatic Concentrations of Baclofen and Acamprosate

To evaluate the overall exposure of the patients to the drugs, baclofenand acamprosate plasmatic concentration has been measured at the end ofthe study (V4), in the morning just before drug administration (on anempty stomach, assumed to be the lowest drug concentration) and twohours after the dosing, which roughly corresponds to the Tmax of thedrugs (assumed to be the highest concentration). Actual plasmaticconcentration of the drugs to which the patient is exposed thusoscillates during the treatment between these lowest and highest drugconcentrations.

The plasmatic concentration of a given drug can be determined using anymethod well-known by biopharmacists. Briefly, an LC-MS/MS analysis hasbeen performed on samples with the material detailed in Table 8, but anyequivalent material can be used.

TABLE 8 acamprosate quantification baclofen quantification ChromatographShimadzu LC-20AD liquid Shimadzu LC-20AB liquid chromatography systemchromatography system SIL-20AC-HT autosampler SIL-20AC autosamplerMatrix C18 stationary phase Phenyl-hexyl stationary phase Mass API5500(AB-Sciex) A TSQ Quantum Ultra spectrometer mass (Thermo ElectronCorporation) Electrospray Negative Positive Ionisation mode ReferenceAcamprosate calcium Baclofen standard Internal Acamprosate-D12 calciumBaclofen-D4 standard trihydrate

Watson® LIMS 7.2 (Thermo Electron, Philadelphia, Pa.) software was usedfor regression, calculation, and statistical calculations.

Analyst 1.5.2 (AB-Sciex) and LCQuan 2.5 (Thermo Electron Corporation)software was used for LC-MS/MS instrument control, data acquisition andintegration. Any other software can be used as a function of the massspectrometer to be used.

Samples are human plasma samples collected during the clinical trial.Blank plasma samples were used to prepare calibration.

For acamprosate quantification, sampling volume was 100 μl and proteinshave been precipitated, and for baclofen, sampling volume was 200 μl andsamples were submitted to solid phase extraction.

Mini Mental State Examination (MMSE) Score

The MMSE is widely used to assess the cognitive functions and the mnesicabilities [65] in the frame of the diagnosis of AD and of the assessmentof the severity of the disease. The test comprises a series of 30questions addressing 5 different areas (orientation, registration,attention calculation, recall, and language) with the final score beinggraded out of 30 points. A final score below or equal to 26 pointscorresponds to a dementia diagnosis. Normal scores range from 30 to 26,whereas a score between 26 and 20 indicates mild dementia, a scorebetween 19 and 10 indicates moderate dementia, and a score below 10 isconsidered as indicating severe dementia. A variation of 2 points of thescore is usually considered as clinically relevant.

ADAS Cog Score

ADAS cognitive subscale deals with memory, language, construction andpraxis orientation and is commonly considered as a standard for theevaluation of mild to moderate AD patients in the clinical trials. Itconsists of 11 tasks which measure the disturbances of memory, language,praxis, attention and other cognitive abilities which are often referredto as the core symptoms of AD. It generally shows good test-retest andinter-rater reliability and performs satisfactorily against moredetailed measures of cognitive function.

Score range varies from 0 to 70. The more mistakes made, the higher thescore. A normal score is defined as a score lower than 10. A 70-pointscore is a sign of severe dementia [66].

a) Combination Therapies Improve Cognitive Performances of Mild ADPatients.

Results presented herein were obtained from an intermediate analysis ofthe ADAS cog data for the 24 first subjects to complete the study whowere treated by doses 1 or 2.

As shown in FIG. 26, the evolution of performances in the ADAS cog testsall along the study shows a clear correlation with the CDR design of thestudy, either when considering the whole set of patients (i.e., whateverthe dosage, FIG. 26), or considering separately patients treated withdose 1 or dose 2 (not shown). It clearly appears that treatment withbaclofen-acamprosate during the challenge and re-challenge phasesresults in an improvement of the subject's cognitive performances at theend of both phases when compared to those measured at the beginning ofeach phase. This positive effect of the treatment is emphasized by theclear worsening of the score during the de-challenge phase, wherein aplacebo is administered.

A strongly significant improvement of performances of patients in theADAS cog test is observed between V1 and V2 (p<0.01). The three-monthstudy led finally to a global significant improvement of patientcondition despite the dividing de-challenge phase (p<0.05).

b) Baclofen-Acamprosate Combination Acts on the ElectrophysiologicalFeatures of Cognition in Mild AD Patients.

Alterations in brain functions can be detected by electrophysiologicaltechniques such as electroencephalography (EEG). Event relatedpotentials (ERPs) are observed within the EEG recordings in response toan experimental condition, and represent the corresponding activatedcognitive phenomena such as perception, attention, decision-makingprocess, answering, memory process, language, etc. ERP characteristicsand their components can vary as a function of various factors such asstimulus relevance, task performed, lesions of the nervous system, theuse of drugs and so on.

ERPs have been found to be altered in patients suffering from AD,vascular dementia or dementia associated with Parkinsonian symptoms.Numerous research studies have helped to establish ERPs as a usefulcognitive biomarker for the diagnosis of dementia, tracking diseaseprogression, and evaluating the pro-cognitive effect of therapeutics.More particularly ERP measures allow pointing out alteration ofcognitive function at an early stage, and can contribute to thediagnosis of AD with good sensitivity and specificity.

The most frequently recorded potential in clinical practice is P300which comprises P3 and N2 subcomponents:

-   -   P3 (or late positive complex) is a large centroparietal        positivity in the ERP that occurs with a latency of        approximately 300 ms after a discordant stimulus. P3 can be        divided in the two subcomponents P3a and P3b. P3a is generally        considered to be related to the degree of focal attention        whereas P3b is supposed to index the working memory update. P3        amplitude notably refers to selective attention stimulus        occurrence probability motivation and vigilance.    -   N2 is a negative wave which precedes P3 and which might be        linked to the detection of the target stimulus and be the        reflection of the selective attention processes coming into        action.

Turning back to AD, a P3b latency increase and amplitude decrease arethe most consensual objective parameters observed in AD patients. It isuseful for the monitoring of AD progression, and even for assessing ADtreatment response. Generally speaking, latency refers to the timenecessary for the making of a decision and amplitude refers to thedifficulty of the task, probability of occurrence of the stimulus, oremotional state.

Recording of ERPs

ERP assessment is done at visits V1-V4 of the above clinical trial.

Recording of cognitive ERPs is done according to the auditory oddballparadigm. Patients lay down on an “examination bed,” with eyes opened,in a soundproof, darkened room. Tones (60 dB SPL, 100-ms duration) werepresented binaurally through a headset up to a total of 150 stimuli.Patients were instructed to identify the odd 2000 Hz high-pitchedstimuli (target sounds), which had a 20% occurrence probability amongthe standard 1000 Hz low-pitched stimuli (common sounds). The cognitivetask required paying attention to the odd stimuli and counting them. Thestimulus order of appearance was random and there was at least a 1140-msgap between each stimulus. Three tests were recorded with a 2-minutepause followed by repeated instructions. The test was stopped once the90 (3×30) target stimuli have been played out and the patient was askedto give out his/her count of the oddest sounds. Separate averaging ofsingle records corresponding to frequent and rare stimuli were processedonline. Grand average of the evoked potentials was calculated from thethree trials for Fz (frontal vertex), Cz (Central vertex) and Pz(Parietal vertex) electrodes.

Evoked potential amplitudes were measured relative to the prestimulusbaseline from the records collected at Fz, Cz and Pz:

-   -   P3 was the most positive wave after N2 between 279 and 440 ms.    -   N100 wave was the most negative peak in the range 75 to 150 ms.    -   N200 wave was the most negative peak between 196 and 300 ms.

In Alzheimer's disease latencies are hugely delayed and amplitudes arereduced.

Latencies and amplitudes of the P3a and P3b subcomponents of P3 and N200were taken from the signals recorded from Fz, Cz and Pz electrodes andused for statistical analyses. Delta of amplitudes between N2 and P3aand delta of amplitudes between N2 and P3b were also calculated duringstatistical analyses. ASCII-format files were also recorded for PCAstatistical analyses.

All these 24 latencies/amplitudes/deltas-of-amplitudes parameters andthe PCA analysis were assessed for each period (challenge, de-challenge,re-challenge) and compared across all these 3 periods.

The analysis was computed with R. All quantitative parameters of theERPs were presented in terms of mean, standard deviation, median,extreme values, Q1, Q3, number of patients and missing data.

Case Study: Baclofen-Acamprosate Combinatorial Treatment SignificantlyCorrects ERP Features in an AD Subject

Results presented in FIG. 27 represent ERP measures gathered from allthree electrodes all along the CDR study for patient 601.

Patient 601 is a 74-year-old female Caucasian subject with no knownantecedent of familial dementia. She was diagnosed as suffering fromprobable AD since June 2012, and presents a moderate hypotrophy of thehippocampus together with a global cerebral hypotrophy in neuroimaging,which ascertains the diagnosis. At V0 she obtained a MMSE score of 24She was administered with the low dose (dose 1) of the combination.

ERPs obtained at V1 for this patient are characterized by a substantialdelay of P3, around 900 ins for this subject (FIG. 27).

As soon as V2, a significant correction of both the delay (a significantdecrease) and the amplitude (a significant enhancement) of the P3b waveis noticed. This positive effect of the mix seems to be maintained evenduring the de-challenge phase: P3b at V3 is found shifted to the leftwhen compared to the one of V2 and also presents a greater amplitude.Improvement of latency is still observed at V4 at the end of there-challenge phase. These results suggest that the mix is efficient inimproving working memory update in AD patients. Noteworthy, animprovement is also observed regarding the P3A and N2 components, whichare more related to the attention processes.

Hence, the baclofen-acamprosate combination is efficient in correctingthe alterations of brain electrophysiological functions which areobserved in mild AD subjects.

c) Conclusion—Clinical Trial

Results of clinical trials performed in humans show the effectiveness ofcombinations of the invention in treating AD and related disorders asshown by the observed protection against memory process degradation bothin a chemically induced amnesia model and in patients diagnosed withmild AD. The correction of memory processes in patients is asserted byelectrophysiology data that show a correction in brain functioning upontreatment in mild AD patients.

A great variability is commonly observed in plasmatic concentrations ofa given drug in response to the same dosing regimen in differentsubjects. This can be due notably to variations in the efficiency ofmetabolism of drugs within each patient. Despite this, it is noteworthythat during the clinical trial patients have been exposed to plasmaticconcentrations of drugs of the same order of magnitude as theconcentrations of baclofen and acamprosate that have been shown tosimultaneously protect neurons from death and protect synaptic andendothelial functions (Table 9).

TABLE 9 Baclofen Acamprosate** (ng/ml) (ng/ml) In vitro ranges for asimultaneous 17.1-213.7 0.058-0.725 protection of neurons, synaptic andendothelial functions (FIG. 23) Plasmatic ranges observed in patients57.6-261.4 0.58-1.20 after one month of treatment* *The lower valuecorresponds to the mean concentration observed in patients throughclinical trial after one month of treatment on an empty stomach, justbefore administration of the composition; the higher value correspondsto the mean concentration of the drugs observed at Tmax. **Acamprosatecalcium

D. Treatment of Age-Associated Memory Impairment.

Memory is known as one of the earliest cognitive functions to declinethrough the aging process in humans and rodents [67, 68].

Inventors have evaluated the efficacy of the compositions of theinvention in treating AD-related diseases such as age-associated memoryimpairment. Working memory performances were evaluated through theT-maze alternation test in treated and non-treated animals (roughly thesame test as spontaneous alternation in part I.B)1)a)).

Animals—Treatment

24-month-old C57L/6J male mice were used. Mice were held on a reversedlight cycle. Each experiment was performed between 8 am and 3 pm and,therefore, under red light conditions.

Mice were administered by gavage, twice daily with either thecombination of the invention (acamprosate 0.2 mg/kg+baclofen 3 mg/kg) orthe vehicle solution all along the experiment. Working memoryperformances of treated and non-treated animals were assayed 17 weeksand 29 weeks after the beginning of the treatment.

On the day of the test, animals were treated 2 hours before the test.

Experimental Procedure

The T-maze apparatus is made of gray Plexiglas with a main stem (55 cmlong×10 cm wide×20 cm high) and two arms (30 cm long×10 cm wide×20 cmhigh) positioned at a 90-degree angle relative to the main stem. A startbox (15 cm long×10 cm wide) is separated from the main stem by a slidingdoor. Sliding doors are also provided to close specific arms during theforced-choice alternation task.

The experimental protocol consists of one single session, which startswith 1 “forced-choice” trial, followed by 14 “free-choice” trials. Inthe first “forced-choice” trial, the animal is confined 5 s in the startarm and then released while either the left or right goal arm is blockedby closing the sliding door. Then the animal explores the open arm andreturns to the start arm. At this point, the animal has completed theforced-choice trial. Immediately after the return of the animal to thestart position, the left or right goal door is opened and the animal isallowed to freely choose between the left and right goal arm (“freechoice” trials). Each time that the animal has chosen a goal arm, theopposite arm is closed in order to oblige the animal to return to thestart arm. Once the animal returns to the start arm, all goal doors areopened to allow another round of free choice trial begins. The animal isconsidered as entered in a choice arm when it places its four paws inthe arm. A session is terminated and the animal is removed from the mazeas soon as 14 free-choice trials have been performed or 15 min haveelapsed, whatever event occurs first.

The apparatus was cleaned between each animal using alcohol (70°). Urineand feces were removed from the maze.

During the trials, animal handling and the visibility of the operatorwere minimized as much as possible.

Calculation and Statistical Analysis

The percent spontaneous alternation was calculated as the number ofspontaneous alternations divided by the number of free-choice trials. Analternation is defined as a succession of 2 different arms overconsecutive choices (e.g., the sequence right-left-right represents 2alternations).

Analysis of variance (ANOVA) was performed on the results. Dunnett'stest was applied to determine significance of differences.

Results

As shown in FIG. 28, composition of the invention significantly improved(by more than 50%) memory of aged mice as soon as 17 weeks after thebeginning of the treatment. A significant improvement is also observedafter 29 weeks of treatment (not shown).

Hence compositions of the invention are also efficient in counteractingAD-related disorders such as age-associated memory impairment.

II. Treatment of Diseases Related to Glutamate Toxicity

A. Prevention of Glutamate Toxicity on Neuronal Cells In Vitro

In this further set of experiments, candidate compounds have been testedfor their ability to prevent or reduce the toxic effects of glutamatetoxicity on neuronal cells. Glutamate toxicity is involved in thepathogenesis of neurological diseases or disorders such as multiplesclerosis, Alzheimer's Disease, frontotemporal dementia, amyotrophiclateral sclerosis, Parkinson's Disease, Huntington's Disease,neuropathies, alcoholism or alcohol withdrawal, or spinal cord injury.The drugs are first tested individually, followed by assays for theircombinatorial action.

Methods

The efficacy of drug combinations of the invention is assessed onprimary cortical neuron cells. The protocol which is used in theseassays is the same as described in section I.A.2 above.

Glutamate Toxicity Assays

The neuroprotective effect of compounds is assessed by quantification ofthe neurite network (neurofilament immunostaining (NF), whichspecifically reveals the glutamatergic neurons).

After 12 days of neuron culture, drugs of the candidate combinations aresolved in culture medium (+0.1% DMSO). Candidate combinations are thenpre-incubated with neurons for 1 hour before the glutamate injury. Onehour after incubation, glutamate is added for 20 min, to a finalconcentration of 40 μM, in the presence of the candidate combinations,in order to avoid further drug dilutions. At the end of the incubation,the medium is changed with medium with the candidate combination butwithout glutamate. The culture is fixed 24 hours after glutamate injuryMK801 (Dizocilpinehydrogen maleate, 77086-22-7-20 μM) is used as apositive control.

After permeabilization with saponin (Sigma), cells are blocked for 2 hwith PBS containing 10% goat serum, then the cells are incubated withmouse monoclonal primary antibody against neurofilament antibody (NF,Sigma). This antibody is revealed with Alexa Fluor 488 goat anti-mouseIgG.

Nuclei of cells are labeled by a fluorescent marker (Hoechst solution,SIGMA), and neurite network quantified. Six wells per condition are usedto assess neuronal survival in 3 different cultures.

Results

The combination of baclofen-acamprosate gives a protective effectagainst glutamate toxicity for cortical neuronal cells. As exemplifiedin FIG. 15, combinations of the invention strongly protect neurons fromglutamate toxicity under the experimental conditions described above. Itis noteworthy that an effective protection is noticed using drugconcentrations at which the drugs used alone have a lower protectiveeffect. Combination of baclofen and acamprosate induces an improvementof more than 200% compared to acamprosate alone and more than 47%compared to baclofen alone.

B. Improvement of Other Disorders Related to Glutamate ExcitoxicityUsing Combinations of the Invention

The above-mentioned in vitro protective effect against glutamatetoxicity of drugs and drug combinations of the invention, combined withthe protective effects exemplified herein in several AD models, promptedthe inventors to test these drugs and combinations in some models ofother diseases in the pathogenesis of which glutamate toxicity is alsoinvolved, such as MS, ALS and neuropathic pain.

1. Protective Effect of Combinations in an In Vivo Model of MultipleSclerosis.

A model in which myelin-oligodendrocyte glycoprotein-immunized(MOG-immunized) mice develop chronic progressive EAE is used todemonstrate the beneficial effect of compositions of the invention inmultiple sclerosis treatment.

Animals and Chemicals

C57L/6J female mice (8 weeks old) are purchased from Janvier (France);after two weeks of habituation, female mice (10 weeks old) developchronic paralysis after immunization with MOG (Myelin OligodendrocyteGlycoprotein) peptide. The experimental encephalomyelitis is inducedwith the Hooke Kit MOG₃₅₋₅₅/CFA Emulsion PTX (Pertussis toxin) for EAEInduction (EK-0110, EK-0115; Hooke Laboratories). The control kit isCK-0115 (Hooke Laboratories).

Experimental Procedure

Experimental encephalomyelitis is induced by following procedure:

On day 0, two subcutaneous injections of 0.1 ml each are performed: onein the upper back of the mouse and one in the lower back. Each injectioncontains 100 μg of MOG₃₅₋₅₅ peptide (MEVGWYRSPFSRVVHLYRNGK, SEQ IDNO:1), 200 μg of inactivated Mycobacterium tuberculosis H37Ra and isemulsified in Complete Freund's Adjuvant (CFA) (Hooke Laboratories). Theemulsion provides antigen needed to expand and differentiateMOG-specific autoimmune T cells.

Two intraperitoneal injections of 500 ng of pertussis toxin in PBS(Hooke kit) are performed 2 hours (Day 0) and 24 hours (Day 1) after theMOG injection. Pertussis toxin enhances EAE development by providingadditional adjuvant.

Mice develop EAE 8 days after immunization and stay chronicallyparalyzed for the duration of the experiment. After the immunization,mice are daily observed for clinical symptoms in a blind procedure.Animals are kept in a conventional pathogen-free facility and allexperiments are carried out in accordance with guidelines prescribed by,and are approved by, the standing local committee of bioethics.

Experimental Groups and Drug Treatment:

Groups of female mice as disclosed are homogenized by weight before theimmunization:

-   -   Control group: vehicle injection in the same conditions of EAE        mice (from Day −1 to Day 28, placebo is given daily),    -   EAE group: MOG injection (Day 0)+pertussis toxin injections (Day        0 and 1)−from Day −1 to Day 28, placebo is given orally daily,    -   EAE+positive control: MOG injection (Day 0)+pertussis toxin        injections (Day 0 and 1)−from Day −1 to Day 28, dexamethazone is        given orally daily, and    -   EAE+treatment group: MOG injection (Day 0)+pertussis toxin        injections (Day 0 and 1). The treatments start one day before        immunization and last until Day 28.

Treatments are applied into two divided doses (i.e., bid).

The clinical scores are measured at Days0-5-8-9-12-14-16-19-21-23-26-28.

Statistical software (Statsoft Inc) is utilized throughout forstatistical analysis. ANOVA analysis and Student's t-test are employedto analyse clinical disease score. P<0.05 is considered significant.

Delays of disease occurrence, clinical score and delay of death havebeen compared between each group to the reference “immu” group withKaplan-Meier curves and a Cox model (R package “survival”). Resultingp-values are unilateral and test the hypothesis to be better than thereference “immu” group.

The total clinical score is composed of the tail score, the hind limbscore, the fore limb score and the bladder score described as below:

Tail Score:

Score = 0 A normal mouse holds its tail erect when moving. Score = 1 Ifthe extremity of the tail is flaccid with a tendency to fall. Score = 2If the tail is completely flaccid and drags on the table.

Hind Limbs Score:

Score = 0 A normal mouse has an energetic walk and doesn't drag its pawsScore = 1 Either one of the following tests is positive: A - Flip test:while holding the tail between thumb and index finger, flip the animalon its back and observe the time it takes to right itself. A healthymouse will turn itself immediately. A delay suggests hind-limb weakness.B - Place the mouse on the wire cage top and observe as it crosses fromone side to the other. If one or both limbs frequently slip between thebars we consider that there is a partial paralysis. Score = 2 Bothprevious tests are positive. Score = 3 One or both hind limbs show signsof paralysis but some movements are preserved; for example: the animalcan grasp and hold on to the underside of the wire cage top for a shortmoment before letting go. Score = 4 When both hind legs are paralyzedand the mouse drags them when moving.

Fore Limbs Score:

Score = 0 A normal mouse uses its front paws actively for grasping andwalking and holds its head erect. Score = 1 Walking is possible butdifficult due to a weakness in one or both of the paws, for example, thefront paws are considered weak when the mouse has difficulty graspingthe underside of the wire top cage. Another sign of weakness is headdrooping. Score = 2 When one forelimb is paralyzed (impossibility tograsp and the mouse turns around the paralyzed limb). At this time thehead has also lost much of its muscle tone. Score = 3 Mouse cannot move,and food and water are unattainable.

Bladder Score:

Score = 0 A normal mouse has full control of its bladder. Score = 1 Amouse is considered incontinent when its lower body is soaked withurine.

The global score for each animal is determined by the addition of allthe above mentioned categories. The maximum score for live animals is10.

Results: Combination Therapies are Efficient in a MS Model

A significant improvement of global clinical score is observed in“EAE+treatment group” mice for the baclofen and acamprosate combination.

The combination of baclofen (30 mg/kg/day) and acamprosate (2 mg/kg/day)induced a significant protective effect against the development ofchronic progressive EAE and hence confirmed the beneficial effect of thecomposition in multiple sclerosis treatment (FIG. 18). With more than30% reduction of the symptoms, the results clearly show that thecombination induces a significant reduction of disease development fromDay 13. This result confirms the remarkable positive effect of thebaclofen-acamprosate combination on neuronal protection includingdemyelination and its implications.

Taken together, these results show that this combination enableseffective protection of neurons against many stresses involved in thedevelopment of neurological disease such as β amyloid, BBB breakdown,glutamate excitotoxicity or demyelination.

2) Protective Effects of Combinations in Models of ALS.

The effect of combination therapies according to the present inventionon ALS have been demonstrated in vitro, in a co-culture model, and invivo, in a mouse model of ALS. Protocols and results are presented inthis section.

a) Protective Effect Against Glutamate Toxicity in Primary Cultures ofNerve-Muscle Co-Culture

Primary Cocultures of Nerve and Muscle Cells

Human muscle is prepared according to a previously described method fromportions of a biopsy of a healthy patient [69]. Muscle cells areestablished from dissociated cells (10,000 cells per well), plated ingelatin-coated 0.1% on 48-well plates and grown in a proliferatingmedium consisting of a mix of MEM medium and M199 medium.

Immediately after satellite cell fusion, whole transverse slices of13-day-old rat Wistar embryos' spinal cords with dorsal root ganglia(DRG) attached are placed on the muscle monolayer, 1 explant per well(in center area). DRG are necessary to achieve a good ratio ofinnervations. Innervated cultures are maintained in mixed medium. After24 h in the usual co-culture, neuritis are observed growing out of thespinal cord explants. They make contacts with myotubes and induce thefirst contractions after 8 days. Quickly thereafter, innervated musclefibres located in proximity to the spinal cord explants are virtuallycontinuously contracting. Innervated fibres are morphologically andspatially distinct from the non-innervated ones and could easily bedistinguished from them.

One co-culture is done (6 wells per condition).

Glutamate Injury

On day 27, co-cultures are incubated with candidate compounds orRiluzole one hour before glutamate intoxication (60 μM) for 20 min.Then, co-cultures are washed and candidate compounds or Riluzole areadded for an additional 48 h. After this incubation time, unfixedcocultures are incubated with α-bungarotoxin coupled with Alexa 488 at aconcentration of 500 nmol/L for 15 min at room temperature. Then,co-cultures are fixed by PFA for 20 min at room temperature. Afterpermeabilization with 0.1% of saponin, co-cultures are incubated withanti-neurofilament antibody (NF).

These antibodies are detected with Alexa Fluor 568 goat anti-mouse IgG(Molecular Probes). Nuclei of neurons are labeled by a fluorescentmarker (Hoechst solution).

Endpoints are (1) Total neurite length, (2) Number of motor units, and(3) Total motor unit area, which are indicative of motor neuron survivaland functionality.

For each condition, 2×10 pictures per well are taken using InCellAnalyzer™1000 (GE Healthcare) with 20× magnification. All the images aretaken in the same conditions.

Results

The baclofen and acamprosate combination effectively protects motorneurons and motor units in the coculture model.

b) Combination Therapies are Efficient in ALS Mouse Model

Experiments are performed on male mice. Transgenic maleB6SJL-Tg(SOD1)2Gur/J mice and their control (respectively SN2726 andSN2297 from Jackson Laboratories, Ben Harbor, USA, distributed byCharles River in France) are chosen in this set of experiments to mimicALS.

Diseased mice express the SOD1-G93A transgene, designed with a mutanthuman SOD1 gene (a single amino acid substitution of glycine to alanineat codon 93) driven by its endogenous human SOD1 promoter. Control miceexpress the control human SOD1 gene.

Randomisation of the Animals

The group assignation and the randomisation of the animals are based onbody weight; for each group, the randomisation is done one day beforethe first treatment

Drug Administration

Mice are dosed with candidate drug treatment diluted in a vehicle fromthe 60^(th) day after birth till death. Diluted solutions of drugcandidates are prepared with water at room temperature just before thebeginning of the administration.

In Drinking Water:

Riluzole is added in drinking water at a final concentration of 6 mg/ml(adjusted to each group's mean body weight) in 5% cyclodextrin. As amouse drinks about 5 ml/day, the estimated administrated dose is 30mg/kg/day, which is a dose that was shown to increase the survival ofmice.

Cyclodextrin is used as vehicle at the final concentration of 5%,diluted in water at room temperature from stock solution (cyclodextrin20%).

Oral Administration (Per Os):

-   -   Drug combinations are administrated per os, daily.    -   Cyclodextrin is used as a vehicle at the final concentration of        5%, diluted in water at room temperature from stock solution        (cyclodextrin 20%).

Clinical Observation

The clinical observation of each mouse is performed daily, from thefirst day of treatment (60 days of age) until death (or sacrifice).Clinical observation consists of studying behavioural tests: onset ofparalysis, “loss of splay”, “loss of righting reflex”, and general gaitobservation:

-   -   Onset of paralysis: The observation consists of paralysis        observation of each limb. Onset of paralysis corresponds to the        day of the first signs of paralysis.    -   The loss of splay test consists of tremors or shaking        notification and the position of hind limb (hanging or splaying        out) when the mouse is suspended by the tail.    -   The loss of righting reflex test evaluates the ability of the        mouse to right itself within 30 sec of being turned on either        side. The righting reflex is lost when the mouse is unable to        right itself. The loss of righting reflex determines the end        stage of disease, the mouse unable to right itself is        euthanized.

Results: Combination Therapies are Efficient in ALS In Vivo Model

An improvement of the disease is observed for the diseased animalstreated with for the baclofen and acamprosate combination.

3. Protective Effect of Combinations in Oxaliplatin-Induced Neuropathyas an In Vivo Model for Neuropathic Pain.

Combinatorial therapies of the present invention are tested in vivo, insuitable models of peripheral neuropathy, i.e., an acute model ofoxaliplatin-induced neuropathy and a chronic model ofoxaliplatin-induced neuropathy. The animals, protocols and results arepresented in this section.

Animal Husbandry

Sprague-Dawley rats (CERJ, France), weighing 150-175 g at the beginningof the experiment of the oxaliplatin treatment (Do) are used. Animalsare housed in a limited access animal facility in a temperature (19.5°C.-24.5° C.) and relative humidity (45%-65%) controlled room with a 12h-light/dark cycle, with ad libitum access to standard pelletedlaboratory chow and water throughout the study. Animals are housed 4 or5 per cage and a one week-acclimation period is observed before anytesting.

Experimental Design

Five following groups of rats are used in all experiments:

Control Groups:

Group 1: vehicle of oxaliplatin (distilled water), i.p./Vehicle ofcandidate combination(s) (distilled water), p.o. daily.

Group 2: oxaliplatin (distilled water), i.p./Vehicle of candidatecombination(s) (distilled water), p.o. daily.

Group 3: oxaliplatin 3 mg/kg i.p./single drug in distilled water, p.o.daily×9.

Tested Composition Groups:

Group 4: oxaliplatin 3 mg/kg i.p./candidate combination(s) in distilledwater, p.o. daily×9.

Group 5: oxaliplatin 3 mg/kg i.p./gabapentin (100 mg/kg) in distilledwater, p.o. on testing days (i.e. D₁ & D₈).

Vehicle and test items are delivered daily from D−1 to D7 (the daybefore the last testing day) whereas gabapentin is administered ontesting days (120 minutes before the test).

All treatments are administered in a coded and random order whenpossible. Doses are expressed in terms of free active substance.

Neuropathy Induction

Acute neuropathy is induced by a single intraperitoneal injection ofoxaliplatin (3 mg/kg).

Chronic peripheral neuropathy is induced by repeated intraperitonealinjections of oxaliplatin (3 mg/kg, i.p.) on days 0, 2, 4 and 7 (CD=12mg/kg, i.p.). Chronic neuropathy in humans is cumulative as well and ismost commonly seen in patients who have received total doses ofoxaliplatin > or =540 mg/m² which corresponds to ˜15 mg/kg as acumulative dose in rats [70].

The oxaliplatin-induced painful neuropathy in rat reproduces the painsymptoms in oxaliplatin-treated patients:

-   -   The thermal hyperalgesia is the earliest symptom. It can be        measured with the acetone test or with the tail-immersion test.    -   The mechanical hyperalgesia appears later. It can be quantified        with the Von Frey test or the paw pressure test.

Animal Dosing and Testing

All drug combinations are administered from the day before the firstintraperitoneal injection of oxaliplatin 3 mg/kg (D−1) and pursued dailyorally until D7. During the testing days (i.e., D1 and D7), the drugcombinations are administered after the test. Animals from thereference-treated group (gabapentin) are dosed only during the testingdays.

Acetone Test

Cold allodynia is assessed using the acetone test by measuring theresponses to thermal non-nociceptive stimulation on D1 (around 24 hafter the first injection of oxaliplatin 3 mg/kg (acute effect ofoxaliplatin), and D8 (chronic effect of oxaliplatin).

In the acetone test, latency of hindpaw withdrawal is measured afterapplication of a drop of acetone to the plantar surface of both hindpaws(reaction time) and the intensity of the response is scored (coldscore). Reaction time to the cooling effect of acetone is measuredwithin 20 sec (cut-off) after acetone application. Responses to acetoneare also graded by the following 4-point scale: 0 (no response); 1(quick withdrawal, flick of the paw); 2 (prolonged withdrawal or markedflicking of the paw); 3 (repeated flicking of the paw with licking orbiting).

For each experimental group, results are expressed as:

-   -   The reaction time, defined as the time expressed in sec required        to elicit paw reaction (mean of 6 measures for each rat together        t SEM).    -   The cumulative cold score, defined as the sum of the 6 scores        for each rat together±SEM. The minimum score is 0 (no response        to any of the 6 trials) and the maximum possible score is 18        (repeated flicking and licking or biting of paws on each of the        six trials).

Statistical Analyses

Student test, unilateral, type 3 is performed. The significance level isset as p<0.05; all the groups are compared to the diseased+vehicle group(oxaliplatin-treated group). Means and standard error means are shown onthe figures.

Results

Oxaliplatin induced a significant decrease in reaction time of pawwithdrawal after acetone application (diseased group+vehicle) during thetime course. This decrease is progressive and significant from Day 1(acute model of oxaliplatin-induced neuropathy) to Day 8 (chronic model)as compared to the vehicle group.

Anti-allodynic effect in acute model and chronic model ofoxaliplatin-induced neuropathy: the baclofen and acamprosate combinationis tested in both models of oxaliplatin-induced neuropathy. It induces asignificant decrease in the cumulative cold score and a significantincrease in reaction time as compared to the oxaliplatin-vehicle treatedgroup. In conclusion, this drug combination protects from chronic andacute neuropathy.

REFERENCES

-   1 Crook R, Verkkoniemi A, Perez-Tur J, Mehta N, Baker M, Houlden H,    Farrer M, Hutton M, Lincoln S, Hardy J, Gwinn K, Somer M, Pactau A,    Kalimo H, Ylikoski R, Poyhonen M, Kucera S & Haltia M (1998) A    variant of Alzheimer's disease with spastic paraparesis and unusual    plaques due to deletion of exon 9 of presenilin 1. Nat. Med. 4,    452-5-   2 Houlden H, Baker M, McGowan E, Lewis P, Hutton M, Crook R, Wood N    W, Kumar-Singh S, Geddes J, Swash M, Scaravilli F, Holton J L,    Lashley T, Tomita T, Hashimoto T, Verkkoniemi A, Kalimo H, Somer M,    Paetau A, Martin J J, Van Broeckhoven C, Golde T, Hardy J, Haltia M    & Revesz T (2000) Variant Alzheimer's disease with spastic    paraparesis and cotton wool plaques is caused by PS-1 mutations that    lead to exceptionally high amyloid-beta concentrations. Ann. Neurol.    48, 806-8.-   3 Kwok J B, Taddei K, Hallupp M, Fisher C, Brooks W S, Broe G A,    Hardy J, Fulham M J, Nicholson G A, Stell R, St George Hyslop P H,    Fraser P E, Kakulas B, Clarnette R, Relkin N, Gandy S E, Schofield P    R & Martins R N (1997) Two novel (M233T and R278T) presenilin-1    mutations in early-onset Alzheimer's disease pedigrees and    preliminary evidence for association of presenilin-1 mutations with    a novel phenotype. Neuroreport 8, 1537-42.-   4 Verkkoniemi A, Kalimo H, Paetau A, Somer M, Iwatsubo T, Hardy J &    Haltia M (2001) Variant Alzheimer disease with spastic paraparesis:    neuropathological phenotype. J. Neuropathol. Exp. Neurol. 60,    483-92.-   5 Citron M (2004) Strategies for disease modification in Alzheimer's    disease. Nat. Rev. Neurosci. 5, 677-85.-   6 Suh Y-H & Checler F (2002) Amyloid precursor protein, presenilins,    and alpha-synuclein: molecular pathogenesis and pharmacological    applications in Alzheimer's disease. Pharmacol. Rev. 54, 469-525.-   7 Blacker D, Albert M S, Bassett S S, Go R C, Harrell L E & Folstein    M F (1994) Reliability and validity of NINCDS-ADRDA criteria for    Alzheimer's disease. The National Institute of Mental Health    Genetics Initiative. Arch. Neurol. 51, 1198-204.-   8 Rossor M N, Fox N C, Freeborough P A & Harvey R J (1996) Clinical    features of sporadic and familial Alzheimer's disease.    Neurodegeneration 5, 393-7.-   9 Glenner G G, Wong C W, Quaranta V & Eanes E D (1984) The amyloid    deposits in Alzheimer's disease: their nature and pathogenesis.    Appl. Pathol. 2, 357-69.-   10 Ballatore C, Lee V M-Y & Trojanowski J Q (2007) Tau-mediated    neurodegeneration in Alzheimer's disease and related disorders. Nat.    Rev. Neurosci. 8, 663-72.-   11 DiLuca M, Bell K F S & Claudio Cuello A (2006) Altered synaptic    function in Alzheimer's disease. Eur. J. Pharmacol. 545, 11-21.-   12 Hardy J A & Higgins G A (1992) Alzheimer's disease: the amyloid    cascade hypothesis. Science 256, 184-5.-   13 Braak H & Braak E (1991) Neuropathological staging of    Alzheimer-related changes. Acta Neuropathol. 82, 239-59.-   14 Golde T E (2005) The Abeta hypothesis: leading us to    rationally-designed therapeutic strategies for the treatment or    prevention of Alzheimer disease. Brain Pathol. 15, 84-7.-   15 Hardy J & Selkoe D J (2002) The amyloid hypothesis of Alzheimer's    disease: progress and problems on the road to therapeutics. Science    297, 353-6.-   16 Selkoe D J (2000) The genetics and molecular pathology of    Alzheimer's disease: roles of amyloid and the presenilins. Neurol.    Clin. 18, 903-22.-   17 Zlokovic B V (2008) The blood-brain barrier in health and chronic    neurodegenerative disorders. Neuron 57, 178-201.-   18 Budd Haeberlein S L & Lipton S A (2009) Excitotoxicity in    neurodegenerative disease. In Encyclopedia of neuroscience (Squire L    R, ed), pp. 77-86. Elsevier.-   19 Hughes J R (2009) Alcohol withdrawal seizures. Epilepsy Rehav.    15, 92-7.-   20 Kim A H, Kerchner G A & Choi D W (2002) CNS Neuroprotection    (F. W. Marcoux and D. W. Choi, eds.) Springer Berlin Heidelberg,    Berlin, Heidelberg.-   21 Hama A & Sagen J (2011) Antinociceptive effect of riluzole in    rats with neuropathic spinal cord injury pain. J. Neurotraunma 28,    127-34.-   22 McGleenon B M, Dynan K B & Passmore Aβ (1999)    Acetylcholinesterase inhibitors in Alzheimer's disease. Br. J. Clin.    Pharmacol. 48, 471-480.-   23 Parsons C G, Danysz W & Quack G (1999) Memantine is a clinically    well tolerated N-methyl-D-aspartate (NMDA) receptor antagonist—a    review of preclinical data. Neuropharmacology 38, 735-67.-   24 Malgouris C, Bardot F, Daniel M, Pellis F, Rataud J, Uzan A,    Blanchard J C & Laduron P M (1989) Riluzole, a novel antiglutamate,    prevents memory loss and hippocampal neuronal damage in ischemic    gerbils. J. Neurosci. 9, 3720-7.-   25 Wahl F, Allix M. Plotkine M & Boulu R G (1993) Effect of riluzole    on focal cerebral ischemia in rats. Eur. J. Pharmacol. 230, 209-14.-   26 Wahl F, Renou E, Mary V & Stutzmann J M (1997) Riluzole reduces    brain lesions and improves neurological function in rats after a    traumatic brain injury. Brain Res. 756, 247-55.-   27 Ettmayer P, Amidon G L, Clement B & Testa B (2004) Lessons    learned from marketed and investigational prodrugs. J. Med Chem. 47,    2393-404.-   28 Beaumont K, Webster R, Gardner I & Dack K (2003) Design of ester    prodrugs to enhance oral absorption of poorly permeable compounds:    challenges to the discovery scientist. Curr. Drug Metab. 4, 461-85.-   29 Heimbach T, Oh D M, Li L Y, Rodriguez-Hornedo N, Garcia G &    Fleisher D (2003) Enzyme-mediated precipitation of parent drugs from    their phosphate prodrugs. Int. J. Pharm. 261, 81-92.-   30 Yang C Y, Dantzig A H & Pidgeon C (1999) Intestinal peptide    transport systems and oral drug availability. Pharm. Res. 16,    1331-43.-   31 Steffansen B, Nielsen C U, Brodin B, Eriksson A H, Andersen R &    Frokjaer S (2004) Intestinal solute carriers: an overview of trends    and strategies for improving oral drug absorption. Eur. J. Pharm.    Sci. 21, 3-16.-   32 Stella V J (2007) Prodrugs: challenges and rewards. (A. Press and    Springer, eds.) Springer Singapore Pte. Limited, New York.-   33 Wermuth C G (2003) Designing prodrugs and bioprecusrors. In The    Practice of Medicinal Chemistry (Hardbound, ed), 2nd ed, pp. 561-585    Academic Press.-   34 Pezron I, Mitra A K, Duvvuri S & Tirucherai G S (2002) Prodrug    strategies in nasal drug delivery. Expert Opin. Ther. Pat. 12,    331-340.-   35 Stella V J (2004) Prodrugs as therapeutics. Expert Opin. Ther.    Pat. 14, 277-280.-   36 Stella V J & Nti-Addae K W (2007) Prodrug strategies to overcome    poor water solubility. Adv. Drug Deliv. Rev. 59, 677-94.-   37 Higuchi T & Stella V J (1975) Pro-drugs as Novel Drug Delivery    System, ACS Sympos American Chemical Society, Washington, D.C.-   38 Roche E B (1977) Design of biopharmaceutical properties through    prodrugs and analogs: a symposium, American P The Academy,    Washington, D.C.-   39 Lal R, Sukbuntherng J, Tai E H L, Upadhyay S, Yao F, Warren M S,    Luo W, Bu L, Nguyen S, Zamora J, Peng G, Dias T, Bao Y, Ludwikow M,    Phan T, Scheuerman R A, Yan H, Gao M, Wu Q Q, Annamalai T, Raillard    S P, Koller K, Gallop M A & Cundy K C (2009) Arbaclofen placarbil, a    novel R-baclofen prodrug: improved absorption, distribution,    metabolism, and elimination properties compared with R-baclofen. J.    Pharmacol. Exp. Ther. 330, 911-21.-   40 Xu F, Peng G, Phan T, Dilip U, Chen J L, Chernov-Rogan T, Zhang    X, Grindstaff K, Annamalai T, Koller K, Gallop M A & Wustrow D    J (2011) Discovery of a novel potent GABA(B) receptor agonist.    Bioorg. Med. Chem. Lett. 21, 6582-5.-   41 Leach A R & Gillet V J An Introduction to Chemoinformatics    (Springer-Verlag New York Inc, ed.).-   42 Rahman S A, Bashton M, Holliday G L, Schrader R & Thornton J    M (2009) Small Molecule Subgraph Detector (SMSD) toolkit. J.    Cheminform. 1, 12.-   43 Stahl H & Wermuth C G (2011) Pharmaceutical salts: Properties,    selection, and use. 2nd ed (Wiley-VCH, ed.).-   44 Hanafi R, Mosad S, Abouzid K, Niess R & Spahn-Langguth H (2011)    Baclofen ester and carbamate prodrug candidates: a simultaneous    chromatographic assay, resolution optimized with DryLab. J. Pharm.    Biomed. Anal. 56, 569-76.-   45 Raboisson P, Breitholtz-Emanuelsson A, Dahllöf H, Edwards L,    Heaton W L, Isaac M, Jarvie K, Kers A, Minidis A B E, Nordmark A,    Sheehan S M, Slassi A, Ström P, Terelius Y, Wensbo D, Wilson J M,    Xin T & McLeod D A (2012) Discovery and characterization of AZD9272    and AZD6538-Two novel mGluR5 negative allosteric modulators selected    for clinical development. Bioorg. Med. Chem. Lett. 22, 6974-9.-   46 Suzuki G, Kimura T, Satow A, Kaneko N, Fukuda J, Hikichi H, Sakai    N, Maehara S, Kawagoe-Takaki H, Hata M, Azuma T, Ito S, Kawamoto H &    Ohta H (2007) Pharmacological characterization of a new, orally    active and potent allosteric metabotropic glutamate receptor 1    antagonist,    4-[1-(2-fluoropyridin-3-yl)-5-methyl-1H-1,2,3-triazol-4-yl]-N-isopropyl-N-methyl-3,6-dihydropyridine-1    (2H)-carboxamide (FTIDC). J. Pharmacol. Exp. Ther. 321, 1144-53.-   47 Kohara A, Nagakura Y, Kiso T, Toya T, Watabiki T, Tamura S,    Shitaka Y, Itahana H & Okada M (2007) Antinociceptive profile of a    selective metabotropic glutamate receptor 1 antagonist YM-230888 in    chronic pain rodent models. Eur. J. Pharmacol. 571, 8-16.-   48 Dekundy A, Gravius A, Hechenberger M, Pietraszek M, Nagel J,    Tober C, van der Elst M, Mela F, Parsons C G & Danysz W (2011)    Pharmacological characterization of MRZ-8676, a novel negative    allosteric modulator of subtype 5 metabotropic glutamate receptors    (mGluR5): focus on L: -DOPA-induced dyskinesia. J. Neural Transm.    118, 1703-16.-   49 Goeldner C, Ballard T M, Knoflach F, Wichmann J, Gatti S &    Umbricht D (2013) Cognitive impairment in major depression and the    mGlu2 receptor as a therapeutic target. Neuropharmacology 64,    337-46.-   50 Jaeschke G, Wettstein J G, Nordquist R E & Spooren W (2008) mGlu5    receptor antagonists and their therapeutic potential. Expert Opin.    Ther. Pat. 18, 123-142.-   51 Carroll F1 (2008) Antagonists at metabotropic glutamate receptor    subtype 5: structure activity relationships and therapeutic    potential for addiction. Ann. N.Y. Acad Sci. 1141, 221-32.-   52 Emmitte K A (2013) mGlu5 negative allosteric modulators: a patent    review (2010-2012). Expert Opin. Ther. Pat. 23, 393-408.-   53 Froestl W, Muhs A & Pfeifer A (2012) Cognitive enhancers    (nootropics). Part 1: drugs interacting with receptors. J.    Alzheimers. Dis. 32, 793-887.-   54 Hemstapat K, Da Costa H, Nong Y, Brady A E, Luo Q, Niswender C M,    Tamagnan G D & Conn P J (2007) A novel family of potent negative    allosteric modulators of group II metabotropic glutamate    receptors. J. Pharmacol. Exp. Ther. 322, 254-64.-   55 Maksay G, Nemes P, Vincze Z & Biró T (2008) Synthesis of    (nor)tropeine (di)esters and allosteric modulation of glycine    receptor binding. Bioorg. Med. Chem. 16, 2086-92.-   56 Yevenes G E & Zeilhofer H U (2011) Allosteric modulation of    glycine receptors. Br. J. Pharmacol. 164, 224-36.-   57 Paris D, Ait-Ghezala G, Mathura V S. Patel N, Quadros A, Laporte    V & Mullan M (2005) Anti-angiogenic activity of the mutant Dutch    A(beta) peptide on human brain microvascular endothelial cells.    Brain Res. Mol. Brain Res. 136, 212-30.-   58 Singer C A, Figueroa-Masot X A, Batchelor R H & Dorsa D M (1999)    The mitogen-activated protein kinase pathway mediates estrogen    neuroprotection after glutamate toxicity in primary cortical    neurons. J. Neurosci. 19, 2455-63.-   59 Harrison N L (1990) On the presynaptic action of baclofen at    inhibitory synapses between cultured rat hippocampal neurones. J.    Physiol. 422, 433-46.-   60 LOEWE S (1953) The problem of synergism and antagonism of    combined drugs Arzneimittelforschung. 3, 285-90.-   61 Grabovsky Y & Tallarida R J (2004) Isobolographic analysis for    combinations of a full and partial agonist: curved isoboles. J.    Pharmacol. Exp. Ther. 310, 981-6.-   62 BLISS C I (1939) THE TOXICITY OF POISONS APPLIED JOINTLY 1. Ann.    Appl. Biol. 26, 585-615.-   63 Meunier J, Ieni J & Maurice T (2006) The anti-amnesic and    neuroprotective effects of donepezil against amyloid beta25-35    peptide-induced toxicity in mice involve an interaction with the    sigma1 receptor. Br. J. Pharmacol. 149, 998-1012.-   64 Snyder P J, Bednar M M, Cromer J R & Maruff P (2005) Reversal of    scopolamine-induced deficits with a single dose of donepezil, an    acetylcholinesterase inhibitor. Alzheimers. Dement. 1, 126-35.-   65 Folstein M F, Folstein S E & McHugh P R (1975) “Mini-mental    state”. A practical method for grading the cognitive state of    patients for the clinician. J. Psychiatr. Res. 12, 189-98.-   66 Gool W A van (2000) Het effect van rivastigmine bij de ziekte van    Alzheimer: houdt rivastigmine het alledaagse gewoon?    Geneesmiddelenbulletin 34, 17-22.-   67 Barnes C A Aging and the physiology of spatial memory. Neurobiol.    Aging 9, 563-8.-   68 Dember W N & Fowler H (1958) Spontaneous alternation behavior.    Psychol. Bull. 55, 412-28.-   69 Braun S, Croizat B, Lagrange M C, Warter J M & Poindron P (1996)    Neurotrophins increase motoneurons' ability to innervate skeletal    muscle fibers in rat spinal cord—human muscle cocultures. J. Neurol.    Sci. 136, 17-23.-   70 Cersosimo R J (2005) Oxaliplatin-associated neuropathy: a review.    Ann. Pharmacother. 39, 128-35.

We claim:
 1. A composition comprising (i) baclofen, (ii) acamprosate,and (iii) idalopirdine, or pharmaceutically acceptable salts,derivatives or prodrugs thereof.
 2. The composition of claim 1, whereinthe compounds are in admixture with a pharmaceutically acceptablecarrier or excipient.
 3. The composition of claim 1, which comprises adose of baclofen of less than 150 mg.
 4. The composition of claim 1,which comprises a dose of acamprosate of less than 1000 mg.
 5. Thecomposition of claim 1, which comprises a dose between 0.4 mg and 50 mgof acamprosate and between 6 and 15 mg of baclofen.
 6. The compositionof claim 1, which comprises baclofen, acamprosate and idalopirdine asthe only active agents.
 7. The composition of claim 1, which is a solidformulation suitable for oral administration.
 8. A method for treatingAlzheimer in a human subject in need thereof, comprising administeringto said subject and effective amount of baclofen, acamprosate andidalopirdine, or pharmaceutical acceptable salt(s) or derivative(s)thereof.
 9. The method of claim 8, wherein the compounds are inadmixture with a pharmaceutically acceptable carrier or excipient. 10.The method of claim 8, wherein baclofen, or the pharmaceuticallyacceptable salt or derivative thereof, is administered at a dose of lessthan 150 mg.
 11. The method of claim 8, wherein acamprosate, or thepharmaceutically acceptable salt or derivative thereof, is administeredat a dose of less than 1000 mg.
 12. The method of claim 8, whereinacamprosate, or the pharmaceutically acceptable salt or derivativethereof, is administered at a dose between 0.4 mg and 50 mg, andbaclofen, or the pharmaceutically acceptable salt or derivative thereof,is administered at a dose between 6 mg and 15 mg, twice daily.
 13. Themethod of claim 8, wherein baclofen, acamprosate and idalopirdine, orthe pharmaceutically acceptable salt(s) or derivative(s) thereof, arethe only agents administered for treating Alzheimer disease.
 14. Themethod of claim 8, wherein baclofen, acamprosate and idalopirdine, orthe pharmaceutically acceptable salt(s) or derivative(s) thereof, areformulated or administered together, separately or sequentially.
 15. Themethod of claim 8, wherein baclofen, acamprosate and idalopirdine, orthe pharmaceutically acceptable salt(s) or derivative(s) thereof, areadministered orally.